Urine mass spectrometry in patients with hepatocellular carcinoma, liver cirrhosis and healthy persons

Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world and
the third cause of cancer related mortality. In many patients HCC occurs
against a background of chronic liver disease, and in a majority cirrhosis is
present. Currently surveillance program schedules for patients with cirrhosis
include alpha-fetoprotein (AFP) blood level determination and liver imaging.
This surveillance strategy has obvious disadvantages related to the sensitivity
and specificity of these tests, especially for detection of early HCC. It would
be of clinical relevance to detect biomarkers that will optimize the positive
predictive value of screening tests.
Human biological fluids contain various types of biomarkers that are found to
be specific and
sensitive reporters of diseases. Human urine plays a central role in clinical
diagnostics and
urine is a sample that is easily obtained. In a recently performed urine mass
spectrometry pilot study, we identified metabolites and natural occurring
peptides (possible new biomarkers) specific for the presence of hepatocellular
carcinoma. We conducted a pilot study for the discovery of specific natural
occurring peptides and metabolites in the urine of patients with hepatocellular
carcinoma. We collected urine of 10 female patients with confirmed
hepatocellular carcinoma. As control groups we selected female patients with
colorectal liver metastases, benign liver adenomas (adenomas) and healthy
persons (kidney donors). More than 31980 different masses can be found of which
186 could be identified as known peptides (Roest et al. EuroKUP WG/MC meeting
March 2010, Rotterdam). From the 31980 masses detected by the mass spectrometry
11 individual masses were found specific in patients with HCC by comparison
analysis using Progenesis (statistical program for mass spectrometry analysis).
With the availability of a large cohort of patients at risk for the development
of HCC (included in the surveillance program of the Department of
Hepato-gastroenterology) and the expertise with mass spectrometry at the
Erasmus MC we have the unique opportunity to continue the study to determine
the relevance of biomarkers for the early detection of HCC.

To determine metabolites and natural occurring peptides in urine by mass
spectrometry in patients with hepatocellular carcinoma. This study is designed
as a validation study; in a smaller study a spectrum of metabolites and natural
occurring peptides have been identified in urine of patients with HCC and
without HCC. However, with the data from the pilot study we cannot discriminate
between biomarkers related to cirrhosis and to hepatocellular carcinoma. As
identification of biomarkers predicting the early presence of hepatocellular
carcinoma in cirrhotic livers would be of great clinical relevance additional
urine analysis using mass spectrometry is indicated to determine a potential
successful test of a new biomarker signature.

Stage 1: Validation
In our recently performed urine mass spectrometry pilot study, metabolites and
natural
occurring peptides are identified which are specific for the presence of
hepatocellular carcinoma. For our validation study we will screen for the 11
specific masses in an expand number of patients with and without HCC, to
identify a set biomarkers ( 3 to 5 metabolites or natural occurring peptides)
that can be used as classifier with the highest sensitivity and specificity.
We expect this number to be sufficient to correct for confounders such as
cirrhosis, hepatitis B viral or hepatitis C viral infection which are the most
important risk factors for HCC. In order to evaluate the sensitivity and
specificity of the classifier we will include a control group of 60 patients
without HCC and running the surveillance program. In addition, a group of
healthy people (kidney donors) (n=60) will be included as reference. The
collected urine samples will be analyzed by Orbitrap mass spectrometry (Thermo
Fisher Scientific). The comparison analysis will be performed by software tools
developed for nanoliquid chromatography MS comparisons (Progenesis, Non Linear
Dynamics, UK).

Stage 2: Effect of treatment on classifier
To optimize the classifier (identified specific metabolites/natural occurring
peptides) and to expand clinical application. Patients with HCC will be
followed after treatment in order to investigate a decrease or disappearance of
the identified metabolites. In case of HCC recurrence we will collect a urine
sample to investigate whether the identified metabolites can be detected.

The blood sample collection is part of the regulier work-up of patients
visiting the outdoor clinic. Urine collection will not case extra risks.