Treatment outcome according to genetic tumour alterations and clinical characteristics in digestive high-grade neuroendocrine neoplasms.

BACKGROUND: Chemotherapy has limited efficacy in advanced digestive high-grade neuroendocrine neoplasms (HG-NEN) and prognosis is dismal. Predictive markers for palliative chemotherapy are lacking, and prognostic markers are limited. METHODS: Digestive HG-NEN patients (n = 229) were prospectively included 2013-2017. Pathological re-assessment revealed 188 neuroendocrine carcinomas (NEC) and 41 neuroendocrine tumours (NET G3). Tumour-DNA was sequenced across 360 cancer-related genes, assessing mutations (mut) and copy number alterations. We linked sequencing results to clinical information and explored potential markers for first-line chemotherapy efficacy and survival. RESULTS: In NEC given cis/carboplatin and etoposide (PE), TP53mut predicted inferior response rate in multivariate analyses (p = 0.009) and no BRAFmut NEC showed response. In overall assessment of PE-treated NEC, no genetic alterations were prognostic for OS. For small-cell NEC, TP53mut were associated with longer OS (p = 0.011) and RB1 deletions predicted lack of immediate-progression (p = 0.003). In non-small cell NEC, APC mut were associated with immediate-progression and shorter PFS (p = 0.008/p = 0.004). For NET G3, ATRXmut, ARID1A- and ERS1 deletions were associated with shorter PFS. CONCLUSION: Correlations between genetic alterations and response/immediate-progression to PE were frequent in NEC but affected PFS or OS only when subdividing for cell-type. The classification of digestive NEC into large- and small-cell seems therefore molecularly and clinically relevant.

Single cell-resolved study of advanced murine MASH reveals a homeostatic pericyte signaling module.

BACKGROUND & AIMS: Metabolic dysfunction-associated steatohepatitis (MASH) is linked to insulin resistance and type 2 diabetes and marked by hepatic inflammation, microvascular dysfunction, and fibrosis, impairing liver function and aggravating metabolic derangements. The liver homeostatic interactions disrupted in MASH are still poorly understood. We aimed to elucidate the plasticity and changing interactions of non-parenchymal cells associated with advanced MASH. METHODS: We characterized a diet-induced mouse model of advanced MASH at single-cell resolution and validated findings by assaying chromatin accessibility, bioimaging murine and human livers, and via functional experiments in vivo and in vitro. RESULTS: The fibrogenic activation of hepatic stellate cells (HSCs) led to deterioration of a signaling module consisting of the bile acid receptor NR1H4/FXR and HSC-specific GS-protein-coupled receptors (GSPCRs) capable of preserving stellate cell quiescence. Accompanying HSC activation, we further observed the attenuation of HSC Gdf2 expression, and a MASH-associated expansion of a CD207-positive macrophage population likely derived from both incoming monocytes and Kupffer cells. CONCLUSION: We conclude that HSC-expressed NR1H4 and GSPCRs of the healthy liver integrate postprandial cues, which sustain HSC quiescence and, through paracrine signals, overall sinusoidal health. Hence HSC activation in MASH not only drives fibrogenesis but may desensitize the hepatic sinusoid to liver homeostatic signals. IMPACT AND IMPLICATIONS: Homeostatic interactions between hepatic cell types and their deterioration in metabolic dysfunction-associated steatohepatitis are poorly characterized. In our current single cell-resolved study of advanced murine metabolic dysfunction-associated steatohepatitis, we identified a quiescence-associated hepatic stellate cell-signaling module with potential to preserve normal sinusoid function. As expression levels of its constituents are conserved in the human liver, stimulation of the identified signaling module is a promising therapeutic strategy to restore sinusoid function in chronic liver disease.

Development, validation, and prognostic evaluation of a risk score for long-term liver-related outcomes in the general population: a multicohort study.

BACKGROUND: Liver cirrhosis is a major cause of death worldwide. Cirrhosis develops after a long asymptomatic period of fibrosis progression, with the diagnosis frequently occurring late, when major complications or cancer develop. Few reliable tools exist for timely identification of individuals at risk of cirrhosis to allow for early intervention. We aimed to develop a novel score to identify individuals at risk for future liver-related outcomes. METHODS: We derived the LiverRisk score from an international prospective cohort of individuals from six countries without known liver disease from the general population, who underwent liver fibrosis assessment by transient elastography. The score included age, sex, and six standard laboratory variables. We created four groups: minimal risk, low risk, medium risk, and high risk according to selected cutoff values of the LiverRisk score (6, 10, and 15). The model's discriminatory accuracy and calibration were externally validated in two prospective cohorts from the general population. Moreover, we ascertained the prognostic value of the score in the prediction of liver-related outcomes in participants without known liver disease with median follow-up of 12 years (UK Biobank cohort). FINDINGS: We included 14 726 participants: 6357 (43·2%) in the derivation cohort, 4370 (29·7%) in the first external validation cohort, and 3999 (27·2%) in the second external validation cohort. The score accurately predicted liver stiffness in the development and external validation cohorts, and was superior to conventional serum biomarkers of fibrosis, as measured by area under the receiver-operating characteristics curve (AUC; 0·83 [95% CI [0·78-0·89]) versus the fibrosis-4 index (FIB-4; 0·68 [0·61-0·75] at 10 kPa). The score was effective in identifying individuals at risk of liver-related mortality, liver-related hospitalisation, and liver cancer, thereby allowing stratification to different risk groups for liver-related outcomes. The hazard ratio for liver-related mortality in the high-risk group was 471 (95% CI 347-641) compared with the minimal risk group, and the overall AUC of the score in predicting 10-year liver-related mortality was 0·90 (0·88-0·91) versus 0.84 (0·82-0·86) for FIB-4. INTERPRETATION: The LiverRisk score, based on simple parameters, predicted liver fibrosis and future development of liver-related outcomes in the general population. The score might allow for stratification of individuals according to liver risk and thus guide preventive care. FUNDING: European Commission under the H20/20 programme; Fondo de Investigación Sanitaria de Salud; Instituto de Salud Carlos III; Spanish Ministry of Economy, Industry, and Competitiveness; the European Regional Development Fund; and the German Ministry of Education and Research (BMBF).

Sphingolipids Are Depleted in Alcohol-Related Liver Fibrosis.

BACKGROUND & AIMS: Alcohol disturbs hepatic lipid synthesis and transport, but the role of lipid dysfunction in alcohol-related liver disease (ALD) is unclear. In this biopsy-controlled, prospective, observational study, we characterized the liver and plasma lipidomes in patients with early ALD. METHODS: We performed mass spectrometry-based lipidomics of paired liver and plasma samples from 315 patients with ALD and of plasma from 51 matched healthy controls. We associated lipid levels with histologic fibrosis, inflammation, and steatosis with correction for multiple testing and adjustment for confounders. We further investigated sphingolipid regulation by means of quantitative real-time polymerase chain reaction sequencing of microRNA, prediction of liver-related events, and tested causality with Mendelian randomization. RESULTS: We detected 198 lipids in the liver and 236 lipids in the circulation from 18 lipid classes. Most sphingolipids (sphingomyelins and ceramides) and phosphocholines were co-down-regulated in both liver and plasma, where lower abundance correlated with higher fibrosis stage. Sphingomyelins showed the most pronounced negative correlation to fibrosis, mirrored by negative correlations in both liver and plasma with hepatic inflammation. Reduced sphingomyelins predicted future liver-related events. This seemed to be characteristic of "pure ALD," as sphingomyelin levels were higher in patients with concomitant metabolic syndrome and ALD/nonalcoholic fatty liver disease overlap. Mendelian randomization in FinnGen and UK Biobanks indicated ALD as the cause of low sphingomyelins, and alcohol use disorder did not correlate with genetic susceptibility to low sphingomyelin levels. CONCLUSIONS: Alcohol-related liver fibrosis is characterized by selective and progressive lipid depletion in liver and blood, particularly sphingomyelins, which also associates with progression to liver-related events.

Screening for Fibrosis Promotes Lifestyle Changes: A Prospective Cohort Study in 4796 Individuals.

BACKGROUND AND AIMS: Early detection of liver fibrosis is believed to promote lifestyle changes. We evaluated self-reported changes in alcohol intake, diet, exercise, and weight after participating in a screening study for liver fibrosis. METHODS: We conducted a prospective screening study of individuals at risk of alcohol-related liver disease (ALD) or metabolic dysfunction-associated steatotic liver disease (MASLD). We provided lifestyle advice to all participants and evaluated lifestyle changes by questionnaires after 1 week and 6 months, with re-examination of a subgroup after 2 years. RESULTS: A total of 1850 at risk of ALD and 2946 at risk of MASLD were included, of whom 383 (8%) were screening positive (transient elastography ≥8 kPa). A total of 84% replied to the 6-month questionnaire. In ALD participants, excessive drinking decreased from 46% to 32% after 6 months. Only 15% reported increased drinking, without differences between screening positive and negative individuals (P = .698). In high-risk drinkers, a positive screening test predicted abstinence or decreased alcohol use after 6 months (odds ratio, 2.45; 95% confidence interval, 1.32-4.57; P = .005). After 2 years, excessive drinking decreased from 52% to 41% in a subgroup of 752 individuals and a positive screening test predicted abstinence or decreased alcohol use after 2 years (odds ratio, 1.84; 95% confidence interval, 1.09-3.11, P = .023). MASLD participants showed similar improvements: 35% improved their diet, 22% exercised more, and 13% reported a weight loss ≥5% after 6 months. CONCLUSIONS: Screening for liver fibrosis is associated with sustained improvements in alcohol consumption, diet, weight, and exercise in at-risk ALD and MASLD. The changes are most pronounced in screening positive participants but not limited to this group.

Circulating TREM2 as a noninvasive diagnostic biomarker for NASH in patients with elevated liver stiffness.

BACKGROUND AND AIMS: Reliable noninvasive biomarkers are an unmet clinical need for the diagnosis of NASH. This study investigates the diagnostic accuracy of the circulating triggering receptor expressed on myeloid cells 2 (plasma TREM2) as a biomarker for NASH in patients with NAFLD and elevated liver stiffness. APPROACH AND RESULTS: We collected cross-sectional, clinical data including liver biopsies from a derivation ( n = 48) and a validation cohort ( n = 170) of patients with elevated liver stiffness measurement (LSM ≥ 8.0 kPa). Patients with NAFLD activity scores (NAS) ≥4 were defined as having NASH. Plasma TREM2 levels were significantly elevated in patients with NASH of the derivation cohort, with an area under the receiver operating characteristics curve (AUROC) of 0.92 (95% confidence interval [CI], 0.84-0.99). In the validation cohort, plasma TREM2 level increased approximately two-fold in patients with NASH, and a strong diagnostic accuracy was confirmed (AUROC, 0.83; 95% CI, 0.77-0.89; p < 0.0001). Plasma TREM2 levels were associated with the individual histologic features of NAS: steatosis, lobular inflammation, and ballooning ( p < 0.0001), but only weakly with fibrosis stages. Dual cutoffs for rule-in and rule-out were explored: a plasma TREM2 level of ≤38 ng/ml was found to be an optimal NASH rule-out cutoff (sensitivity 90%; specificity 52%), whereas a plasma TREM2 level of ≥65 ng/ml was an optimal NASH rule-in cutoff (specificity 89%; sensitivity 54%). CONCLUSIONS: Plasma TREM2 is a plausible individual biomarker that can rule-in or rule-out the presence of NASH with high accuracy and thus has the potential to reduce the need for liver biopsies and to identify patients who are eligible for clinical trials in NASH.

Identification of tumour regression in neoadjuvantly treated pancreatic cancer is based on divergent and nonspecific criteria.

AIMS: Following the increased use of neoadjuvant therapy for pancreatic cancer, grading of tumour regression (TR) has become part of routine diagnostics. However, it suffers from marked interobserver variation, which is mainly ascribed to the subjectivity of the defining criteria of the categories in TR grading systems. We hypothesized that a further cause for the interobserver variation is the use of divergent and nonspecific morphological criteria to identify tumour regression. METHODS AND RESULTS: Twenty treatment-naïve pancreatic cancers and 20 pancreatic cancers treated with neoadjuvant chemotherapy were reviewed by three experienced pancreatic pathologists who, blinded for treatment status, categorized each tumour as treatment-naïve or neoadjuvantly treated, and annotated all tissue areas they considered showing tumour regression. Only 50%-65% of the cases were categorized correctly, and the annotated tissue areas were highly discrepant (only 3%-41% overlap). When the prevalence of various morphological features deemed to indicate TR was compared between treatment-naïve and neoadjuvantly treated tumours, only one pattern, characterized by reduced cancer cell density and prominent stroma affecting a large area of the tumour bed, occurred significantly more frequently, but not exclusively, in the neoadjuvantly treated group. Finally, stromal features, both morphological and biological, were investigated as possible markers for tumour regression, but failed to distinguish TR from native tumour stroma. CONCLUSION: There is considerable divergence in opinion between pathologists when it comes to the identification of tumour regression. Reliable identification of TR is only possible if it is extensive, while lesser degrees of treatment effect cannot be recognized with certainty.

Exploring the prognostic value of circular RNAs in pancreatic ductal adenocarcinoma using genome-wide expression profiling.

BACKGROUND/OBJECTIVES: Median survival of pancreatic ductal adenocarcinoma (PDAC) is around eight months and new prognostic tools are needed. Circular RNAs (circRNAs) have gained interest in different types of cancer. However, only a few studies have evaluated their potential in PDAC. We aimed to identify the most differentially expressed circRNAs in PDAC compared to controls and to explore their potential as prognostic markers. METHODS: Using frozen specimens with PDAC and controls, we performed RNA sequencing and identified 20,440 unique circRNAs. A custom code set of capture- and reporter probes for NanoString nCounter analysis was designed to target 152 circRNAs, based on abundancy, differential expression and a literature study. Expression of these 152 circRNAs was examined in 108 formalin-fixed and paraffin-embedded surgical PDAC specimens and controls. The spatial expression of one of the most promising candidates, ciRS-7 (hsa_circ_0001946), was evaluated by chromogenic in situ hybridization (CISH) using multi-punch tissue microarrays (TMAs) and digital imaging analysis. RESULTS: Based on circRNA expression profiles, we identified different PDAC subclusters. The 30 most differentially expressed circRNAs showed log2 fold changes from -3.43 to 0.94, where circNRIP1 (hsa_circ_0004771), circMBOAT2 (hsa_circ_0007334) and circRUNX1 (hsa_circ_0002360) held significant prognostic value in multivariate analysis. CiRS-7 was absent in PDAC cells but highly expressed in the tumor microenvironment. CONCLUSIONS: We identified several new circRNAs with biomarker potential in surgically treated PDAC, three of which showed an independent prognostic value. We also found that ciRS-7 is absent in cancer cells but abundant in tumor microenvironment and may hold potential as marker of activated stroma.

Effect of Calorie-Unrestricted Low-Carbohydrate, High-Fat Diet Versus High-Carbohydrate, Low-Fat Diet on Type 2 Diabetes and Nonalcoholic Fatty Liver Disease : A Randomized Controlled Trial.

BACKGROUND: It remains unclear if a low-carbohydrate, high-fat (LCHF) diet is a possible treatment strategy for type 2 diabetes mellitus (T2DM), and the effect on nonalcoholic fatty liver disease (NAFLD) has not been investigated. OBJECTIVE: To investigate the effect of a calorie-unrestricted LCHF diet, with no intention of weight loss, on T2DM and NAFLD compared with a high-carbohydrate, low-fat (HCLF) diet. DESIGN: 6-month randomized controlled trial with a 3-month follow-up. (ClinicalTrials.gov: NCT03068078). SETTING: Odense University Hospital in Denmark from November 2016 until June 2020. PARTICIPANTS: 165 participants with T2DM. INTERVENTION: Two calorie-unrestricted diets: LCHF diet with 50 to 60 energy percent (E%) fat, less than 20E% carbohydrates, and 25E% to 30E% proteins and HCLF diet with 50E% to 60E% carbohydrates, 20E% to 30E% fats, and 20E% to 25E% proteins. MEASUREMENTS: Glycemic control, serum lipid levels, metabolic markers, and liver biopsies to assess NAFLD. RESULTS: The mean age was 56 years (SD, 10), and 58% were women. Compared with the HCLF diet, participants on the LCHF diet had greater improvements in hemoglobin A1c (mean difference in change, -6.1 mmol/mol [95% CI, -9.2 to -3.0 mmol/mol] or -0.59% [CI, -0.87% to -0.30%]) and lost more weight (mean difference in change, -3.8 kg [CI, -6.2 to -1.4 kg]). Both groups had higher high-density lipoprotein cholesterol and lower triglycerides at 6 months. Changes in low-density lipoprotein cholesterol were less favorable in the LCHF diet group than in the HCLF diet group (mean difference in change, 0.37 mmol/L [CI, 0.17 to 0.58 mmol/L] or 14.3 mg/dL [CI, 6.6 to 22.4 mg/dL]). No statistically significant between-group changes were detected in the assessment of NAFLD. Changes were not sustained at the 9-month follow-up. LIMITATION: Open-label trial, self-reported adherence, unintended weight loss, and lack of adjustment for multiple comparisons. CONCLUSION: Persons with T2DM on a 6-month, calorie-unrestricted, LCHF diet had greater clinically meaningful improvements in glycemic control and weight compared with those on an HCLF diet, but the changes were not sustained 3 months after intervention. PRIMARY FUNDING SOURCE: Novo Nordisk Foundation.

Prognostic importance of margin clearance in ampullary adenocarcinoma following pancreaticoduodenectomy in a Danish population-based nationwide study.

BACKGROUND: In this Danish nationwide population-based study, we evaluated the prognostically relevant minimum tumour-free margin width following pancreaticoduodenectomy (PD) for ampullary adenocarcinoma (AAC) and evaluated whether certain margins hold independent prognostic information. METHODS: We included 128 patients who underwent PD for AAC from 2015 to 2019. Clinical and pathological data including well-known prognostic factors were retrieved from the Danish Pancreatic Cancer Database. Missing data were obtained by review of pathology reports and re-microscopy of resection specimens. All PD specimens were examined using a standardised pathological protocol including multicolour inking, axial slicing and exact reporting of margin widths. The cohort was dichotomised into involved and uninvolved groups, using different margin clearance definitions (0.5-≥3.0 mm). RESULTS: Following PD for AAC, margin clearance of ≥1 mm was independently associated with improved chance of survival compared with <1 mm (HR: 0.30, 95 % CI: 0.14-0.64 (p = 0.002)). Posterior and anterior margin widths were narrower compared with superior mesenteric artery and vein margins. Posterior margin and anterior surface had isolated prognostic significance in multivariable analysis. CONCLUSION: Following PD for AAC, margin clearance of at least 1 mm is independently associated with improved survival. Our data further indicate that anterior surface and posterior margin hold particular prognostic value.

Phase II study of everolimus and temozolomide as first-line treatment in metastatic high-grade gastroenteropancreatic neuroendocrine neoplasms.

BACKGROUND: The optimal treatment for metastatic high-grade gastroenteropancreatic (GEP) neuroendocrine neoplasms when Ki-67 ≤55% is unknown. A prospective multi-centre phase 2 study was performed to evaluate the efficacy and safety of everolimus and temozolomide as first-line treatment for these patients. METHODS: Patients received everolimus 10 mg daily continuously and temozolomide 150 mg/m2 for 7 days every 2 weeks. Endpoints included response, survival, safety and quality of life (QoL). Histopathological re-evaluation according to the 2019 WHO classification was performed. RESULTS: For 37 eligible patients, the primary endpoint with 65% disease control rate (DCR) at 6 months (m) was reached. The response rate was 30%, the median progression-free survival (PFS) 10.2 months and the median overall survival (OS) 26.4 months. Considering 26 NET G3 patients, 6 months DCR was 77% vs. 22% among nine NEC patients (p = 0.006). PFS was superior for NET G3 vs. NEC (12.6 months vs. 3.4 months, Log-rank-test: p = 0.133, Breslow-test: p < 0.001). OS was significantly better for NET G3 (31.4 months vs. 7.8 months, p = 0.003). Grade 3 and 4 toxicities were reported in 43% and 38%. QoL remained stable during treatment. CONCLUSION: Everolimus and temozolomide may be a treatment option for selected GEP-NET G3 patients including careful monitoring. Toxicity did not compromise QoL. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov (NTC02248012).

Using the ELF test, FIB-4 and NAFLD fibrosis score to screen the population for liver disease.

BACKGROUND & AIMS: There is a need for accurate biomarkers of fibrosis for population screening of alcohol-related and non-alcoholic fatty liver disease (ALD, NAFLD). We compared the performance of the enhanced liver fibrosis (ELF) test to the fibrosis-4 index (FIB-4) and NAFLD fibrosis score (NFS), using transient elastography as the reference standard. METHODS: We prospectively included participants from the general population, and people at risk of ALD or NAFLD. Screening positive participants (TE ≥8 kPa) were offered a liver biopsy. We measured concomitant ELF, FIB-4, and NFS using validated cut-offs: ≥9.8, ≥1.3, ≥-1.45, respectively. RESULTS: We included 3,378 participants (1,973 general population, 953 at risk of ALD, 452 at risk of NAFLD), with a median age of 57 years (IQR: 51-63). Two hundred-and-forty-two were screening positive (3.4% in the general population, 12%/14% who were at-risk of ALD/NAFLD, respectively). Most participants with TE <8 kPa also had ELF <9.8 (88%) despite a poor overall correlation between ELF and TE (Spearman´s rho = 0.207). ELF was associated with significantly fewer false positives (11%) than FIB-4 and NFS (35% and 45%), while retaining a low rate of false negatives (<8%). A screening strategy of FIB-4 followed by ELF in indeterminate cases resulted in false positives in 8%, false negatives in 4% and the correct classification in 88% of cases. We performed a liver biopsy in 155/242 (64%) patients who screened positive, of whom 54 (35%) had advanced fibrosis (≥F3). ELF diagnosed advanced fibrosis with significantly better diagnostic accuracy than FIB-4 and NFS: AUROC 0.85 (95% CI 0.79-0.92) vs. 0.73 (0.64-0.81) and 0.66 (0.57-0.76), respectively. CONCLUSION: The ELF test alone or combined with FIB-4 for liver fibrosis screening in the general population and at-risk groups reduces the number of futile referrals compared to FIB-4 and NFS, without overlooking true cases. IMPACT AND IMPLICATIONS: We need referral pathways that are efficient at detecting advanced fibrosis from alcohol-related and non-alcoholic fatty liver disease in the population, but without causing futile referrals or excessive use of resources. This study indicates that a sequential test strategy of FIB-4 followed by the ELF test in indeterminate cases leads to few patients referred for confirmatory liver stiffness measurement, while retaining a high rate of detected cases, and at low direct costs. This two-step referral pathway could be used by primary care for mass, targeted, or opportunistic screening for liver fibrosis in the population. CLINICAL TRIAL NUMBER: Clinicaltrials.gov number NCT03308916.

A randomized phase II study of full dose gemcitabine versus reduced dose gemcitabine and nab-paclitaxel in vulnerable patients with non-resectable pancreatic cancer (DPCG-01).

BACKGROUND: According to current evidence, the best treatment for fit patients with non-resectable pancreatic cancer (PC) is combination chemotherapy, whereas frail patients are recommended gemcitabine (Gem) monotherapy. Randomized controlled trials in colorectal cancer and a post-hoc analysis of gemcitabine and nab-paclitaxel (GemNab) in PC suggest, however, that reduced dose of combination chemotherapy may be feasible and more efficient compared to monotherapy in frail patients. The aim of this study is to investigate whether reduced dose GemNab is superior to full dose Gem in patients with resectable PC, who are not candidates for full dose combination chemotherapy in first line. METHODS: The Danish Pancreas Cancer Group (DPCG)-01 trial is a national multicenter prospective randomized phase II trial. A total of 100 patients in ECOG performance status 0-2 with non-resectable PC, not candidate for full dose combination chemotherapy in first line, but eligible for full dose Gem, will be included. Patients are randomized 1:1 to either full dose Gem or GemNab in 80% of recommended dose. The primary endpoint is progression-free survival. Secondary endpoints are overall survival, overall response rate, quality of life, toxicity and rate of hospitalizations during treatment. The correlation between blood inflammatory markers, including YKL-40 and IL-6, circulating tumor DNA, and tissue biomarkers of resistance to chemotherapy and outcome will be explored. Finally, the study will include measures of frailty (G8, modified G8, and chair-stand-test) to assess whether scoring would enable a personalized allocation to different treatments or indicates a possibility for interventions. DISCUSSION: Single-drug treatment with Gem has for frail patients with non-resectable PC been the main treatment option for more than thirty years, but the impact on outcome is modest. If improved results and sustained tolerability with reduced dose combination chemotherapy can be shown, this could change the future practice for this increasing group of patients. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT05841420. Secondary Identifying No: N-20210068. EudraCT No: 2021-005067-52. PROTOCOL VERSION: 1.5, 16-MAY-2023.

Validation of scores of PRO-C3 to predict liver-related events in alcohol-related liver disease.

BACKGROUND AND AIMS: Risk prediction in alcohol-related liver disease (ArLD) is an unmet need. We aimed to assess PRO-C3 models to predict liver-related events (LRE) in patients with a history of excessive alcohol use without an established diagnosis of chronic liver disease. METHODS: A prospective cohort study of 462 patients with ArLD, split into a derivation cohort of 221 secondary care patients and a validation cohort of 241 primary care patients. Baseline variables, including fibrogenesis marker PRO-C3, were used to develop a prediction model. Prognostic accuracy was compared to enhanced liver fibrosis (ELF), fibrosis-4-index (FIB-4), transient elastography (TE) and ADAPT. RESULTS: In the derivation and validation cohorts, 67 (30%) and 19 (8%) experienced an LRE during a median follow-up of 5.2 years (IQR: 3.2-6.8) and 4.0 years (IQR: 2.7-5.6). On top of PRO-C3 and ADAPT score, we generated a model (ALPACA) of independent predictors of LREs (PRO-C3, AST/ALT, platelets). ALPACA had high prognostic accuracy with a C-statistic of 0.85 in the derivation cohort, comparable to ELF (0.83) and TE (0.84) and significantly higher than FIB-4 (0.78), PRO-C3 (0.80) and ADAPT (0.81). In the validation cohort, all tests had comparable C-statistics. Compared to low-risk patients (ALPACA ≤11), high-risk patients (>11) had a subhazard ratio for LREs of 12.6 (95% CI 5.9-26.8, p < .001) and higher cumulative incidence (57% vs. 7%, p < .001; derivation cohort). We observed similar subhazard ratio in the validation cohort. CONCLUSIONS: PRO-C3-based scores are reliable tools to predict LREs in ArLD patients and are suitable for risk stratification in primary and secondary care.

Hepatorenal Index by B-Mode Ratio Versus Imaging and Fatty Liver Index to Diagnose Steatosis in Alcohol-Related and Nonalcoholic Fatty Liver Disease.

OBJECTIVES: We aimed to evaluate the accuracy of the hepatorenal index by B-mode ratio to diagnose hepatic steatosis, compared to ultrasound steatosis score, controlled attenuation parameter, and the fatty liver index using histology as the gold standard. METHODS: We prospectively included participants with alcohol-related or nonalcoholic fatty liver disease for same-day noninvasive investigations and liver biopsy. RESULTS: We included 137 participants, 72% male, median age 60 years (53-65) and body mass index 32 kg/m2 (28-38). Eighty percent had steatosis (S0/S1/S2/S3 = 20/37/24/19%). B-mode ratio had moderate diagnostic accuracy for any steatosis (≥S1, area under the receiver operating characteristics curve [AUROC] = 0.79; 95% confidence interval 0.70-0.88), significant steatosis (≥S2, AUROC = 0.76; 0.66-0.85), and severe steatosis (=S3, AUROC = 0.74; 0.62-0.86), independent of disease etiology. The cutoff values to rule-out and rule-in any steatosis were 1.09 and 1.45. While B-mode ratio and controlled attenuation parameter correlated poorly, their diagnostic accuracies were comparable to each other and to ultrasound steatosis scoring. Fatty liver index did not differ from B-mode ratio in detecting any steatosis but had poor accuracy to detect higher steatosis grades. B-mode ratio measurements failed in 12% of patients, compared to 1% for ultrasound steatosis scoring and 2% for controlled attenuation parameter. CONCLUSION: The hepatorenal index by B-mode ratio diagnose steatosis with moderate accuracy in patients with alcohol-related or nonalcoholic fatty liver disease, comparable to B-mode ultrasound steatosis scoring and controlled attenuation parameter. However, its clinical use is limited by a high failure rate.

Prognostic significance of margin clearance in pancreaticoduodenectomy specimens with pancreatic ductal adenocarcinoma in a Danish population-based nationwide study.

BACKGROUND: In this nationwide population-based cohort study, we investigated the overall minimum margin width that is independently associated with improved survival following pancreaticoduodenectomy (PD) for pancreatic ductal adenocarcinoma (PDAC) and evaluated whether certain margins or surfaces hold independent prognostic significance. METHODS: Data from 367 patients who underwent PD for PDAC in the period 2015-2019 were retrieved from the Danish Pancreatic Cancer Database. Missing data were obtained by review of pathology reports and re-microscopy of resection specimens. Surgical specimens were evaluated using a standardised pathological protocol involving multicolour inking, axial slicing and exact reporting of circumferential margin clearances in 0.5 mm increments. RESULTS: When categorised according to margin widths of <0.5, <1.0, <1.5, <2.0, <2.5 and <3.0 mm, R1 resections were detected in 34%, 57%, 75%, 78%, 86% and 87% of cases, respectively. In multivariable analyses, an overall margin clearance of ≥1.5 mm was associated with improved survival compared with a clearance of <1.5 mm (HR 0.70 95% CI 0.51-0.97 (p = 0.031)). When evaluating the margins separately, no margin had independent prognostic significance. CONCLUSION: Margin clearance of at least 1.5 mm was independently associated with improved survival following PD for PDAC.

Metabolic and Genetic Risk Factors Are the Strongest Predictors of Severity of Alcohol-Related Liver Fibrosis.

BACKGROUND & AIMS: Individual risk for developing alcohol-related liver disease (ALD) varies greatly. We hypothesized that metabolic risk factors and genetic polymorphisms predict severity of ALD. METHODS: Biopsy-controlled, cross-sectional study in patients with a history of excessive drinking. We measured the homeostatic model assessment of insulin resistance (HOMA-IR), plasma triglycerides, high- and low-density lipoproteins (HDL, LDL), and total cholesterol. Moreover, we genotyped four single nucleotide polymorphisms in PNPLA3 (rs738409C>G), TM6SF2 (rs58542926C>T), MBOAT7 (rs641738C>T), and HSD17B13 (rs72613567T>TA). We assessed predictors of higher fibrosis stage using multivariable ordered logistic regression. RESULTS: Of 325 included patients, 25% had severe fibrosis or cirrhosis and 59% had HOMA-IR ≥2.5. HOMA-IR increased for each fibrosis stage, while there was a similar decrease in LDL and total cholesterol. Individuals with risk variant PNPLA3 rs738409-G or TM6SF2 rs58542926-T had higher fibrosis stage. In multivariable regression, HOMA-IR ≥2.5 (OR = 3.04, 95% CI 1.90-4.87), LDL <2.60 mmol/L (OR = 2.05, 95% CI 1.33-3.16), TM6SF2 rs58542926-T (OR = 1.99, 95% CI 1.17-3.37), age above 50 years (OR = 1.66, 95% CI 1.03-2.70), and PNPLA3 rs738409-G (OR = 1.54, 95% CI 1.11-2.12) independently predicted higher fibrosis stage. Independent predictors of hepatic inflammatory activity were HOMA-IR, active drinking, age, and PNPLA3 risk variant. Active drinking, elevated triglycerides, and PNPLA3 risk variant predicted steatosis. CONCLUSIONS: Insulin resistance is the strongest predictor of liver fibrosis stage and hepatic inflammation in patients with alcohol-related liver disease. Genetic susceptibility further aggravates this risk. These data highlight the clinical value of detailed metabolic and genetic profiling of patients with excessive alcohol use.

LiverScreen project: study protocol for screening for liver fibrosis in the general population in European countries.

BACKGROUND: The development of liver cirrhosis is usually an asymptomatic process until late stages when complications occur. The potential reversibility of the disease is dependent on early diagnosis of liver fibrosis and timely targeted treatment. Recently, the use of non-invasive tools has been suggested for screening of liver fibrosis, especially in subjects with risk factors for chronic liver disease. Nevertheless, large population-based studies with cost-effectiveness analyses are still lacking to support the widespread use of such tools. The aim of this study is to investigate whether non-invasive liver stiffness measurement in the general population is useful to identify subjects with asymptomatic, advanced chronic liver disease. METHODS: This study aims to include 30,000 subjects from eight European countries. Subjects from the general population aged ≥ 40 years without known liver disease will be invited to participate in the study either through phone calls/letters or through their primary care center. In the first study visit, subjects will undergo bloodwork as well as hepatic fat quantification and liver stiffness measurement (LSM) by vibration-controlled transient elastography. If LSM is ≥ 8 kPa and/or if ALT levels are ≥1.5 x upper limit of normal, subjects will be referred to hospital for further evaluation and consideration of liver biopsy. The primary outcome is the percentage of subjects with LSM ≥ 8kPa. In addition, a health economic evaluation will be performed to assess the cost-effectiveness and budget impact of such an intervention. The project is funded by the European Commission H2020 program. DISCUSSION: This study comes at an especially important time, as the burden of chronic liver diseases is expected to increase in the coming years. There is consequently an urgent need to change our current approach, from diagnosing the disease late when the impact of interventions may be limited to diagnosing the disease earlier, when the patient is asymptomatic and free of complications, and the disease potentially reversible. Ultimately, the LiverScreen study will serve as a basis from which diagnostic pathways can be developed and adapted to the specific socio-economic and healthcare conditions in each country. TRIAL REGISTRATION: This study is registered on Clinicaltrials.gov ( NCT03789825 ).

Prognostic performance of 7 biomarkers compared to liver biopsy in early alcohol-related liver disease.

BACKGROUND & AIMS: Alcohol is the most common cause of liver-related mortality and morbidity. We therefore aimed to assess and compare the prognostic performance of elastography and blood-based markers to predict time to the first liver-related event, severe infection, and all-cause mortality in patients with a history of excess drinking. METHODS: We performed a prospective cohort study in patients with early, compensated alcohol-related liver disease. At baseline, we obtained a liver biopsy, transient elastography (TE), 2-dimensional shear-wave elastography (2D-SWE), enhanced liver fibrosis test (ELF), FibroTest, fibrosis-4 index (FIB-4), non-alcoholic fatty liver fibrosis score (NFS) and Forns index. We compared C-statistics and time-dependent AUC for prognostication. We used validated cut-off points to create 3 risk groups for each test: low, intermediate and high risk. RESULTS: We followed 462 patients for a median of 49 months (IQR 31-70). Median age was 57 years, 76% were males, 20% had advanced fibrosis. Eighty-four patients (18%) developed a liver-related event after a median of 18 months (7-34). TE had the highest prognostic accuracy, with a C-statistic of 0.876, and time-dependent AUC at 5 years of 0.889, comparable to 2D-SWE and ELF. TE, ELF and 2D-SWE outperformed FibroTest, FIB4, NFS, Forns index and biopsy-verified fibrosis stage. Compared to patients with TE <10 kPa, the hazard ratios for liver-related events for TE 10-15 kPa were 8.1 (3.2-20.4), and 27.9 (13.8-56.8) for TE >15 kPa. Periods of excessive drinking during follow-up increased the risk of progressing to liver-related events, except for patients in the low-risk groups. CONCLUSION: TE, ELF and 2D-SWE are highly accurate prognostic markers in patients with alcohol-related liver disease. Easy-to-use cut-offs can distinguish between substantially different risk profiles. LAY SUMMARY: Alcohol is the leading cause of death and illness due to liver disease. In this study, we assessed the ability of biomarkers to predict the risk of developing symptomatic liver disease in patients with early stages of alcohol-related liver disease. We found that several tests accurately predicted the risk of liver-related events such as ascites, esophageal varices and hepatic encephalopathy during an average follow-up of 4.1 years. Liver stiffness measurements by ultrasound elastography and the enhanced liver fibrosis test performed best. By using them, we were able to stratify patients into 3 groups with significantly different risks.

Development and prognostic relevance of a histologic grading and staging system for alcohol-related liver disease.

BACKGROUND & AIMS: The SALVE Histopathology Group (SHG) developed and validated a grading and staging system for the clinical and full histological spectrum of alcohol-related liver disease (ALD) and evaluated its prognostic utility in a multinational cohort of 445 patients. METHODS: SALVE grade was described by semiquantitative scores for steatosis, activity (hepatocellular injury and lobular neutrophils) and cholestasis. The histological diagnosis of steatohepatitis due to ALD (histological ASH, hASH) was based on the presence of hepatocellular ballooning and lobular neutrophils. Fibrosis staging was adapted from the Clinical Research Network staging system for non-alcoholic fatty liver disease and the Laennec staging system and reflects the pattern and extent of ALD fibrosis. There are 7 SALVE fibrosis stages (SFS) ranging from no fibrosis to severe cirrhosis. RESULTS: Interobserver κ-value for each grading and staging parameter was >0.6. In the whole study cohort, long-term outcome was associated with activity grade and cholestasis, as well as cirrhosis with very broad septa (severe cirrhosis) (p <0.001 for all parameters). In decompensated ALD, adverse short-term outcome was associated with activity grade, hASH and cholestasis (p = 0.038, 0.012 and 0.001, respectively), whereas in compensated ALD, hASH and severe fibrosis/cirrhosis were associated with decompensation-free survival (p = 0.011 and 0.001, respectively). On multivariable analysis, severe cirrhosis emerged as an independent histological predictor of long-term survival in the whole study cohort. Severe cirrhosis and hASH were identified as independent predictors of short-term survival in decompensated ALD, and also as independent predictors of decompensation-free survival in compensated ALD. CONCLUSION: The SALVE grading and staging system is a reproducible and prognostically relevant method for the histological assessment of disease activity and fibrosis in ALD. LAY SUMMARY: Patients with alcohol-related liver disease (ALD) may undergo liver biopsy to assess disease severity. We developed a system to classify ALD under the microscope by grading ALD activity and staging the extent of liver scarring. We validated the prognostic performance of this system in 445 patients from 4 European centers.