Introduction

The availability of new drugs for the treatment of patients with metabolic dysfunction-associated steatotic liver disease (MASLD) underlines the need of early predictors of response to such therapies. This study evaluated the impact of 1-year changes in liver stiffness measurement by Vibration-Controlled Transient Elastography (LSM by VCTE), controlled attenuation parameters (CAP), and serum alanine aminotransferase (ALT) on liver outcomes in patients with MASLD.

Aims & Methods

A large multicenter cohort of MASLD patients with LSM≥8kPa and prospective follow-up was enrolled. Liver-related events (LRE), including hepatocellular carcinoma (HCC) and liver decompensation (LD), were evaluated during follow-up. LSM, CAP, and ALT were assessed at baseline and at 1-year follow-up. Cumulative incidence functions and cause-specific Cox regression analyses were performed to correlate 1-year reduction in LSM, CAP, and ALT with the risk of developing LRE, LD and HCC.

Results

In 2443 patients with MASLD and LSM≥8kPa, a 1-year reduction in ALT of 17 IU/L (HR 0.62; 95% CI 0.41-0.95; p=0.02) and a 20% reduction in LSM (HR 0.44; 95% CI 0.29-0.67; p<0.001) were both independently associated with a lower risk of LRE. Similarly, in 1412 patients with MASLD and LSM≥10kPa, a 1-year reduction in ALT of at least 17 IU/L (HR 0.62; 95% CI 0.39-0.98; p=0.04) and a 20% reduction in LSM (HR 0.42; 95% CI 0.27-0.66; p<0.001) were independently associated with a decreased risk of LRE. Comparable results were observed when considering LD separately, but not for HCC. One-year changes in CAP did not predict liver outcomes. When combining 1-year reduction in ALT of 17 IU/L and a 1-year 20% reduction in LSM, the 5-year probabilities of LRE ranged from 2.1% in the best profile to 5% in the worst profile in patients with LSM≥8kPa, and from 3.3% to 8.1% in those with LSM≥10kPa.

Conclusion

One-year reductions in ALT of 17 IU/L and in LSM by 20%, but not changes in CAP, reduce long-term liver outcomes in patients with MASLD, providing suggestion on monitoring therapy effectiveness.

Introduction

Biliary strictures are usually caused by malignancies such as Cholangiocarcinoma (CCA) or Pancreatic Ductal Adenocarcinoma (PDAC). 70% of these neoplasms are diagnosed at advanced stages due to late-onset symptoms, lack of biomarkers, and challenges in obtaining histological samples. Curative surgical resection with adjuvant therapy is the first-line treatment, but 20% of cases are resectable at diagnosis and 80% relapse within 3 years^1,2. The impact of systemic therapies on overall survival remains limited, however new targeted therapies have shown remarkable efficacy in patients with actionable mutations based on tumor genetic profiling. Clinical guidelines advocate multigene panel analysis of tumor tissue prior to or during first-line treatment^3,4, considering that 40% of CCA patients and around 25% of PDAC patients harbor actionable genetic mutations^5,6. When tumor tissue is insufficient, liquid biopsies using cfDNA can be considered³. Liquid biopsy of bile, collected during an Endoscopic Retrograde Cholangiopancreatography (ERCP) in malignant biliary stricture cases, could potentially detect therapeutic targets.

Aims & Methods

This prospective proof-of-concept pilot study aims to assess NGS-based cfDNA analysis in bile as a novel liquid biopsy for detecting genetic mutations, and specifically actionable mutations, compared to tissue NGS.
19 CCA and 9 PDAC patients with malignant biliary stenosis undergoing ERCP in a tertiary hospital (2017-2020) were included. Bile samples were collected during the first ERCP and the Pathological Anatomy department selected tissue samples with ≥40% tumor content. Massive sequencing of DNA libraries was performed using the Pan-Cancer Panel^TM (52-genes) for bile samples, and the Oncomine^TM Comprehensive Assay Panel v3 (161 genes) for tissue.

Results

Concerning CCA patients, bile NGS identified all patients with mutations (19/19) opposite to 84.3% (16/19) of patients in tissue. Bile NGS identified 7 patients (37%) with actionable mutations, none was detected in tissue. We detected 47/49 mutations in bile NGS vs. 22/49 in tissue. High concordance was observed: 91% (20/22) of tissue mutations were also in bile. Regarding actionable mutations considered in ESMO guidelines³; 7 were detected in bile NGS, none detected in tissue. The actionable mutations identified were ERBB2 (5), IDH2 (1), and KRAS^G12C(1).
In the PDAC group, bile NGS identified 89% (8/9) of patients with mutations, compared to 66.6% (6/9) of patients in tissue. Bile NGS identified 5 patients (55%) with actionable mutations vs. 4 (44%) in tissue. Bile NGS detected 16/18 mutations in bile in contrast to 12/18 in tissue. High concordance was observed: 83.3% (10/12) of tissue mutations were also in bile. NGS in bile detected 6 actionable mutations considered in ESMO guidelines^4,; KRAS^G12D (5) and ERBB3, compared to 4 in tissue; KRAS^G12D.

Conclusion

These results highlight the potential of bile liquid biopsy in detecting actionable mutations; bile NGS increased the diagnosis sensibility and the number of mutations detected, compared with tissue NGS, which could allow more patients to be considered for targeted therapies. This approach does not require histological material consumption and collecting bile during an ERCP is simple and secure. In addition, NGS technology is available in all molecular laboratories. We consider that the application of liquid biopsy in bile can be accessible and an effective tool, with direct and immediate clinical implementation, improving precision medicine strategies and the therapeutic management of CCA and PDAC patients with biliary stenosis.

References

1. Banales JM, Marin JJG, Lamarca A, Rodrigues PM, Khan SA, Roberts LR, et al. Cholangiocarcinoma 2020: the next horizon in mechanisms and management. Nat Rev Gastroenterol Hepatol. 2020 Sep;17(9):557-588. Epub 2020 Jun 30.
2.Park W, Chawla A, O’Reilly EM. Pancreatic Cancer: A Review. JAMA. 2021 Sep 7;326(9):851–62.3.
3.Vogel A, Ducreux M; ESMO Guidelines Committee. ESMO Clinical Practice Guideline interim update on the management of biliary tract cancer. ESMO Open. 2025 Jan;10(1):104003. Epub 2024 Dec 17.
4.Conroy T, Pfeiffer P, Vilgrain V, Lamarca A, Seufferlein T, O’Reilly EM, et al. Pancreatic cancer: ESMO Clinical Practice Guideline for diagnosis, treatment and follow-up. Ann Oncol. 2023 Nov;34(11):987–1002.
5. Gilbert TM, Randle L, Quinn M, McGreevy O, O'leary L, Young R, et al. Molecular biology of cholangiocarcinoma and its implications for targeted therapy in patient management. Eur J Surg Oncol. 2025 Feb;51(2):108352. Epub 2024 Apr 17
6. Boileve A, Smolenschi C, Lambert A, Boige V, Tarabay A, Valery M, et al. Role of molecular biology in the management of pancreatic cancer. World J Gastrointest Oncol. 2024 Jul 15;16(7):2902-2914.

Introduction

Primary sclerosing cholangitis (PSC) is a chronic inflammatory disease affecting the biliary epithelium, leading to bile duct strictures and eventually cirrhosis. Liver fibrosis is a well-established predictor of disease outcome in PSC. The Enhanced Liver Fibrosis (ELF) test, which combines three direct serum markers—hyaluronic acid (HA), tissue inhibitor of metalloproteinases-1 (TIMP-1), and amino-terminal pro-peptide of type III pro-collagen (PIIINP)—has been shown to correlate with the Ishak staging of liver histology in PSC. Both the ELF score and its individual components demonstrate moderate diagnostic accuracy in distinguishing advanced fibrosis (AUROC 0.73–0.78) and cirrhosis (AUROC 0.73–0.81) [1].
However, liver fibrosis and cirrhosis in PSC develop as secondary consequences of bile duct strictures and therefore represent late-stage indicators of disease progression. We have previously shown that biliary levels of calprotectin and interleukin-8 (IL-8) can help identify patients at higher risk of developing bile duct strictures. Moreover, our findings indicated that commonly used liver function tests, particularly plasma alkaline phosphatase (P-ALP), lack both sensitivity and specificity for effectively monitoring PSC progression [2].

Aims & Methods

To study: 1) the association of ELF with ERC-score, 2) the predictive value of ELF for bile duct disease progressio assessed by ≥2 increase in ERC score or need for dilatation, 3) the role of ELF-test to predict the overall disease outcome.
We included patients from Helsinki PSC-registry with ELF determined at least once, and ≥2 ERC examinations. End of f/u was 31.12.23. ERC -score was determined as described earlier [3]. ELF ≥9.8 was used as a marker for advanced liver fibrosis.

Results

In total, we included 397 patients. Demographics, laboratory results, and outcomes stratified by ELF levels, are presented in the table. Increase of ERC-score did not differ between groups, but the score at baseline associated with ELF-test (p<0.001). Progression of PSC assessed by increase of ERC score≥2 or dilatation did not correlated with ELF-levels (p=0.11), but ln[P-hyaluronic acid] showed close association with progression of bile duct changes (p<0.001). The probability for LTx increased in a linear way with the increase of ELF-level. (<0.001).

Variables	ELF ≤9.8 N=310	ELF 9.8-11.2 N=62	ELF ≥11.3 N=25	p for linearity
Person years. [Median (IQR) of person years]	2181 [5.6(3.5,11.8)]	414 [5.6(3.5,11.8)]	82 [2.5(1.3,3.8)]	-
PSC duration, mean (SD), years	6.9(5.8)	8.8(6.0)	11.2(8.1)	<0.001
AOM, mean (SD)	1.39(0.46)	1.83(0.56)	2.49(0.67)	<0.001
P-ALP, U/l, median (IQR)	94(74,135)	168(107,250)	317(203,438)	<0.001
P-Bil, µmol/l, median (IQR)	11(8,17)	13(9,18)	29(17,48)	<0.001
Biliary calprotectin, mg/l, median (IQR)	1.3(0.2,15.8)	13.4(0.9,71.0)	22.4(9.1,133.0)	<0.001
Increase of ERC score ≥2, n(%)*	35(11)	9(15)	4(16)	0.35
Increase of ERC score≥2 or dilatation, n(%)*	142(46)	40(65)	20(80)	<0.001
CCA, HCC, GBCA, Death, LTx (Incidence (95% CI), [no])**	15(10 to 21), [32]	60(39 to 89), [25]	220(30 to 347), [18]	<0.001

* Adjusted linearity for age, sex, PSC duration, intra-and extrahepatic disease,and ERC-score at baseline
** per 1000 pyrs. Adjusted for age, sex, PSC duration, intra-and extrahepatic disease.
Abbreviations: AOM, Amsterdam-Oxford model; CCA, cholangiocarcinoma, GB-CA, gallbladder carcinoma; LTx, liver transplantation.

Conclusion

The ELF test correlates with the severity of bile duct disease and the risk of liver transplantation, but it is not suitable for assessing the progression of bile duct changes based on sequential ERC examinations. The role of hyaluronic acid as a surrogate marker of bile duct changes deserves further studies.

References

[1] Thorburn DT et al. Hepatology Communications 2024;8(7):e0467, [2] Färkkilä M et al. JHEP Rep. 2024 Jul 2;6(10):101161, [3] Boyd S, et al. Endoscopy. 2016;48:432-9.