Clarity for patients with a liver adenoma, without a liver biopsy.

a. Substantiate the starting aspect of your application:
In our center we have a relatively large population of patients with liver
adenomas. Liver biopsies are taken from these patients and tested for any
possible mutations. The technique we want to use to analyze circulating DNA
from these patients is also already routinely applied in our center to patients
in follow-up for lung cancer. This combination of circumstances is very unique.
Not only has no attempt been made to date to analyze circulating DNA from
patients with a liver adenoma for mutations in exon 3 of β-catenin. Also, few
centers are well positioned to conduct this study. The risk-bearing aspects are
that this analysis has never been attempted before; that it is unknown whether
there is sufficient adenoma-specific circulating DNA; and whether false
positives will occur if exon 3 mutations are present elsewhere in the patient's
body. This makes the project suitable as a pilot, on the other hand, without
the data from this pilot project, an application for a larger subsidy has
little chance.

b. indicate the innovative nature of the project:
This project aims to collect data that allows patients with liver adenomas to
be discharged from follow-up without a liver biopsy or referred for surgery. We
think we can achieve this by analyzing circulating DNA from these patients for
mutations in exon 3 of CTNNB1 (the gene that codes for the β-catenin protein).
In patients in whom these mutations are demonstrated, resection should take
place; in patients in whom these mutations are excluded, reassurance may be
provided and follow-up may no longer be necessary. This is expected to be an
important cost-saving step.

The question of this project is: can we demonstrate and exclude exon 3
mutations in CTNNB1 by analyzing circulating DNA from patients with a liver
adenoma? The work plan is as follows: we include patients with a liver adenoma
of at least 3 cm in diameter who undergo a diagnostic liver biopsy with
subsequent sequencing of exon 3 of CTNNB1 as part of a currently ongoing study
(gold standard diagnostics). We take a blood sample from these patients. In
this sample we will analyze circulating DNA using the Ion AmpliSeq* HD Library
Kit and analysis with the Ion GeneStudio* S5 system. We will compare the
results of the analysis of the circulating DNA with the results of the analysis
of the liver biopsy (gold standard) to determine whether a liver biopsy can be
omitted in the future.

Liver adenomas usually have a benign course. However, there is a subgroup of
often young patients who have a real clinical risk of malignant deterioration
of the liver adenoma. This concerns patients with a mutation of exon 3 of
CTNNB1 (β-catenin). Of all liver adenomas, approximately 7% have an exon 3
mutation1. Many patients currently remain in follow-up even though they have a
negligible risk of malignant development, which causes concern and increases
the burden on the healthcare system. Recently, as part of an ongoing study in
our center, liver biopsies were taken from patients with a liver adenoma of at
least 3 cm. The sequence of exon 3 was determined in the biopsies to determine
whether an exon 3 mutation is present. When an exon 3 mutation is present,
resection is usually advised. An expectant policy is followed in the other
patients.

A liver biopsy is not without risk. It would therefore be elegant to determine
or exclude a possible exon 3 mutation in peripheral blood. We think this is
possible as this has been shown for mutations in exon 3 of CTNNB1 in three
patients with hepatoblastoma2. The molecular pathology department has also
succeeded in demonstrating TP53 mutations in circulating DNA in high-grade
serous ovarian cancer lesions up to one cm in diameter4. This technique is also
routinely performed in our center to monitor circulating DNA from plasma of
patients with lung cancer.

The question of this project is therefore: can we identify which patients have
an exon 3 mutation in CTNNB1 in the liver adenoma by analyzing circulating DNA
from patients with a liver adenoma? We include patients with a liver adenoma of
at least 3 cm in diameter who undergo a diagnostic liver biopsy with subsequent
sequencing of exon 3 of CTNNB1 (gold standard) as part of a currently ongoing
study. The Gastrostart subsidy allows 30 samples to be analyzed (costs are ~300
euros per sample). We plan to include 15 patients with an exon 3 mutation in
the liver biopsy, and 15 patients with a liver adenoma without exon 3 mutation
as a control group. Analysis of these patients will teach us whether the
technique offers sufficient prospects as a diagnostic test.

We take a blood sample from all patients. In this sample we will analyze
circulating DNA using the Ion AmpliSeq* HD Library Kit and analysis with the
Ion GeneStudio* S5 system 3. We will compare the results of the analysis of the
circulating DNA with the results of the analysis on the liver biopsy. We
consider our study proposal as a serious pilot project. We realize that the
analysis of 30 samples will not sufficiently support an application of this
technique in clinical practice. Follow-up research after a successful pilot
should substantiate our findings in larger cohorts. Because we hypothesize that
the described approach is very cost-effective, as part of the population can be
spared a liver biopsy and possibly be discharged from follow-up and could
therefore avoid many imaging examinations and outpatient examinations, we will
also calculate a cost effectiveness analysis to our pilot study. We have
experience in doing such a cost-effectiveness analysis on a limited amount of
clinical data5.

References:
1 Campani et al. Genetics of Hepatocellular Carcinoma: From Tumor to
Circulating DNA. 2023, Cancers 2023, 15,
817.
2 Kahana-Edwin et al. Exploration of CTNNB1 ctDNA as a putative biomarker for
hepatoblastoma. Pediatric. Blood Cancer 2020 Nov;67(11): e28594.
3 Van Loy et al. Thermo Fisher Scientific, Life Sciences Solutions, 5781 Van
Allen Way, Carlsbad, California, U.S.A. 92008.
4 Vitale et al. TP53 Mutations in Serum Circulating Cell-Free Tumor DNA As
Longitudinal Biomarker for High-Grade Serous Ovarian Cancer. Biomolecules. 2020
Mar; 10(3): 415.
5 Janmaat et al. Cost-Effectiveness of Cetuximab for Advanced Esophageal
Squamous Cell Carcinoma. PLoS One. 2016 Apr 21;11(4):e0153943.

Take an extra tube of blood at the time of an already planned venipuncture.
Very low burden and very low (no) risk.