Non-invasive diagnosis of non-alcoholic steatohepatitis in patients with non-alcoholic fatty liver disease using markers and imaging techniques

Non-alcoholic fatty liver disease (NAFLD) is with 20-30% the most prevalent
liver disorder in Western society and is associated with overweight and obesity
in 50-100%. The majority of patients have simple steatosis, in about 15-30% a
chronic inflammatory state develops that causes non-alcoholic steatohepatitis
(NASH), which leads to an overall increase in morbidity and mortality due to
the progression to fibrosis, cirrhosis and in some cases hepatocellular
carcinoma (HCC). The term NAFLD comprises both simple steatosis and NASH. Most
patients with NAFLD have no or few, mainly aspecific symptoms; and generally
there is a silent progression of simple steatosis to NASH and in the end,
liver-related morbidity and mortality.
To date, liver biopsy is the most sensitive test for detecting and staging
NAFLD, and is the only reliable method for differentiating between NASH and
simple steatosis. However, the procedure of obtaining a liver biopsy is
invasive and associated with patient discomfort, significant complications and
high costs. In addition, liver biopsy is prone to sampling error and inter- and
intra-observer variability, due to the small size of liver biopsy samples. This
method is not suitable for screening large numbers of subjects at risk, or for
follow-up of patients with NASH over time. Further insight into disease
mechanisms and risk factors for NAFLD and in particular NASH is warranted, to
enable early diagnosis, adequate therapy and preventive measures to improve
health status of these individuals. Hence, only subjects at high risk (usually
based upon elevated aminotransferase levels, which is not specific for the
presence of NASH) are biopsied, leading to an underestimation of NASH
prevalence and undertreatment. Accurate and less invasive methods to evaluate
NASH, and NAFLD, are warranted.

The primary objective of this study is to identify non-invasive tools (e.g.
biomarkers and imaging) to diagnose patients with NASH. The secondary objective
is to identify an association between markers and disease severity and to
investigate the effect of a short-term low calorie (CRASH) diet on hepatic
steatosis and inflammation in NAFLD/NASH.

Eligible subjects will be included via the outpatient clinics of the MUMC+,
Zuyderland in Heerlen and the Catharina hospital in Eindhoven. A subset of
eligible subjects have undergone a liver biopsy for clinical reasons. It is
estimated that about 75% of subjects will be asked to undergo a biopsy for
study purposes only. Liver biopsies for study purposes will be combined with a
surgical procedure, e.g. bariatric surgery or cholecystectomy.

Blood, faeces and exhaled air will be collected and a FibroScan (+CAP) will be
performed during a study visit. Furthermore, anthropometric data will be
collected by the research physician, consisting of weight, height and abdominal
and waist circumference. In case a pre-operative low calorie diet is prescribed
as part of standard clinical care (only in the bariatric surgery group) these
measurements will be done before the diet and on the day of the operation.

The participants in the group undergoing liver biopsy during bariatric surgery
will be asked permission to be approached for follow-up measurements 6 months
post-surgery. As they will lose weight, which is associated with improvement of
hepatic steatosis, this enables assessment of possible changes over time. A
routine follow-up visit post-surgery will take place after 6 months. The
follow-up measurements will be combined with this visit, minimizing the burden
for the participant. The measurements will consist of blood, faeces and exhaled
air collection and a FibroScan (+CAP) will be performed during a study visit.
Furthermore, weight, height, BP and abdominal and waist circumference will be
measured.

The measurements, data and sample collections will be planned with the
participant. Blood and faeces collection, exhaled air (VOC) and FibroScan
(+CAP) will be performed. Furthermore, we try to combine study
visits/investigations with outpatient clinical visits.
Side effects of the investigations are a small bruise of taking the blood
samples. Three tubes (21 ml) will be collected for research purposes; if
possible this will be combined with regular blood sampling. Because all
subjects visit the outpatient clinic regularly most of the standard clinical
laboratory investigations (e.g. liver enzymes, albumin, INR, creatinine,
Glucose and HbA1c, insulin, lipid spectrum, ferritin) probably have been
collected for clinical reasons. If not, a maximum of 4 extra tubes (max.17.5 ml
in total) will be collected to determine (part of) the standard clinical
laboratory investigations for research purposes only.
A subset of participants undergoing liver biopsy for study purposes only, are
exposed to certain risks, since liver biopsy is an invasive procedure.
Therefore, any liver biopsies taken for study purposes only, will be combined
with a surgical procedure, e.g. bariatric surgery or cholecystectomy, to
minimize the risks. The biopsy will only take place (during the operation) when
there is written informed consent. The biopsy will be a laparoscopy guided
liver biopsy, not a blind liver biopsy. Possible complications of a liver
biopsy consist of bleeding in the liver and pain in the right upper abdomen of
right shoulder. These complications happen in less than 5% of cases when it
concerns a percutaneous liver biopsy. However, in case of a laparoscopy guided
liver biopsy risks of complications are much lower as patients are anesthetized
and the biopsy is taken by the surgeon while looking at the biopsy side,
before, during and after the biopsy has been taken. Also, the pain related to
the liver biopsy will probably be less distinct, as the patient is anesthetized
during the biopsy procedure and the pain related to the surgical procedure is
probably more prominent. The risk for bleeding is very minimal, as the liver
biopsy is performed at the start of the surgical procedure. The place where the
biopsy is taken can be checked for bleeding, both just after taking the biopsy
and at the end of the surgical procedure, making sure no bleeding has occurred
later on. This minimizes the risk of bleeding complications. During the
surgical procedure a needle and a wedge biopsy will be performed.
The time burden associated with participating in this research is: 1 hour for
the visit to collect samples and perform a physical examination and a FibroScan
(+CAP). In case a pre-operative low calorie diet is prescribed an extra hour is
needed for measurements on the day of the operation, these measurements include
weight measurement, blood, stool and exhaled air collection and a Fibroscan
(+CAP). The observation time post-liver biopsy will be in sync with the
post-operative recovery.
The follow-up visit after 6 months will take 1 hour, to collect samples, to
perform a physical examination and a FibroScan (+CAP).
In case of unexpected findings, awareness of normally unknown pathology may
affect a person*s perception of his own health condition negatively. On the
other hand, early detection is likely to have favourable effects on disease
progression and enable early intervention.
In total subjectin the cholecystectomy group have a time burden of 1 hour and
in the bariatric group maximal 3 hours.