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Hann et al. Urine markers for HCC monitoring

tumor ablation. [12-16] The high HCC recurrence rate patients with a history of HBV infection were studied
can be attributed to (1) incomplete treatment; (2) at the Liver Disease Prevention Center, Division of
micro-metastases within the liver; and (3) de novo Gastroenterology and Hepatology, Thomas Jefferson
lesions. [4,17] With improved assay, combination of alpha University Hospital, Philadelphia. After curative tumor
fetal protein (AFP), lens culinaris agglutinin-reactive ablation, patients were monitored for recurrence by MRI
alpha-fetoprotein (AFP-L3%) and des-gamma- and serum AFP. Urine specimens were prospectively
carboxyprothrombin (DCP) has been claimed sensitive obtained when available. The urine was retrospectively
for HCC surveillance. [18,19] Nonetheless, early detection examined for the presence of the three HCC DNA
of recurrent HCC has been difficult with the currently biomarkers. All patient samples were obtained with
available diagnostic methods and serial imaging. [7-9,20-22] written informed consent and under institutional board
Notably, there are no specific guidelines addressing approval from Thomas Jefferson University Hospital,
how HCC recurrence should be monitored. Magnetic Philadelphia, PA.
resonance imaging (MRI)/computed tomography (CT)
imaging is the gold standard for diagnosis, although Urine DNA analysis
it is expensive and has limited utility in the detection Urine collection, storage, and DNA isolation were
of small tumors (< 2 cm), tumors in the presence carried out with written informed consent from patients
of previously treated lesions (especially from local as described previously. [34,35] DNA from specimens was
ablation), cirrhosis, obesity, and dysplastic nodules. [8,9,20] isolated and fractionated to obtain low molecular weight
Thus, there is an urgent unmet medical need to have a (LMW) urine DNA (< 1 kb size). Bisulfite (BS) treatment
sensitive test for monitoring HCC recurrence. of DNA was performed using the EZ DNA Methylation-
Lightning™ Kit (Zymo Research, Irvine, CA) following
Cancer is a disease of the genome and epigenome, manufacturer’s guidelines. Three DNA modifications,
and detection of the underlying genetic mutations and TP53 249T mutation (TP53m), aberrant promoter
epigenetic modifications in the periphery may allow methylation of GSTP1 (mGSTP1), and aberrant
us to detect cancer early. [23,24] Previously, Su et al. [25-29] promoter methylation of RASSF1A (mRASSF1A),
demonstrated that fragmented cell-free DNA in urine were quantified in duplicate using assays kits, TP53
contains DNA derived from the solid tumors including 249T qPCR kit, mGSTP1 qPCR kit, and mRASSF1A
HCC and colon cancer, if such a tumor is present. qPCR kit (JBS Science Inc., Doylestown, PA), as per
They also demonstrated that cancer-related DNA (both manufacturer specification.
mutated and methylated DNA), including HCC-derived
DNA modifications, could be detected in the urine of RESULTS
patients with cancer. [27,29-31]
To compare the detection of urine DNA markers to the
In this study, we demonstrate the feasibility of early currently available diagnostic methods (serum AFP
detection of recurrent HCC by detecting three and MRI imaging) for the diagnosis of HCC recurrence,
known HCC associated DNA modifications: TP53 urine DNA marker values were measured in a blinded
249T mutation (shortened TP53m), and aberrant fashion and plotted alongside serum AFP at the time
promoter methylation of Glutathione S-transferase pi of each collection (as shown in Figure 1 and described
1 (mGSTP1) and Ras association domain family 1 in “METHODS”). Briefly, urine samples were collected
isoform A (mRASSF1A) genes in urine as compared to prospectively from HCC patients (when available) after
the MRI imaging in a small (n = 10) blinded prospective curative treatment at follow-up visits. The samples were
study. These three DNA markers were chosen because retrospectively analyzed for the HCC DNA biomarkers.
of the availability of sensitive, cell-free DNA suitable For data analysis purposes, we plotted “positive (Pos)”
PCR assays that target frequently altered genes in as the time of confirmed recurrence by MRI, and
major pathways associated with hepatocarcinogenesis. “negative (Neg)” when MRI did not detect recurrence.
They were previously demonstrated to be detectable in Of the 10 patients with > 6 months of monitoring with
body fluids such as blood and urine of patients with urine DNA markers, cases 1-5 had recurrence of HCC
HCC, regardless the level of serum AFP. [29,30,32,33] They confirmed by MRI.
therefore serve as potential biomarkers for HCC.
Recurrent patients had one or more of the three DNA
METHODS markers examined found in urine before or at the time
of MRI diagnosis. One recurrent case (case 5) died of
Patient selection progressive HCC. Case 6 was lost for follow-up during
To explore the potential of the three HCC markers the period of the study. Four patients (cases 7-10) had
for monitoring HCC recurrence in urine, 10 HCC no recurrence confirmed by MRI. Their urine DNA
106 Hepatoma Research ¦ Volume 3 ¦ June 6, 2017