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Hann et al. Urine markers for HCC monitoring

TACE in a 3-year period. Urine was collected every and then shipped to certified laboratories for testing,
6 months starting 4 years after the last TACE. TP53m the urine screening may result in better compliance
mutation was detected in the urine collected on the while not requiring a doctor’s office visit. A larger
second visit and decreased, but remained detectable longitudinal study will be needed to explore the
in the third urine sample as indicated in Figure 1I. MRI application of urine DNA markers in monitoring HCC
suggested a mass in the liver, but the mass was not recurrence. Lastly, the levels of DNA biomarkers in
confirmed as recurrent HCC. The serum AFP levels urine may also be useful to measure effectiveness
were below 20 ng/mL in the period of study. The patient of cancer treatments that induces apoptosis of
has been on antiviral treatment since the diagnosis of tumor cells. We have shown that circulating tumor
HCC. DNA found in urine was mostly from apoptotic tumor
cells. [28,34] The treatment that induce apoptosis should
Case 10 increase the amount of tumor derived DNA deposited
A 66-year-old male with HCC underwent RFA in the blood and secreted into urine. This could be the
followed by resection. He has had no recurrence for circumstance for cases 2, 3 and 4 where an elevated
the past 10 years. Two urine samples were collected mRASSF1A or mGSTP1 marker was detected after
at 8 years (-18) and 9 years (-6) after resection the treatment, suggesting the potential to use urine
[Figure 1J]. Serum AFP is normal, and none of the DNA markers to monitor effectiveness of therapy that
DNA markers were detected until 6 months prior to induces tumor cell apoptosis.
the MRI, when the TP53m and mRASSF1A markers
were elevated [Figure 1J]. TP53m reverted to baseline Finally, HCC is often recognized as being multi-clonal.
and mRASSF1A levels declined 3 months later (-3). At Interestingly, in recurrent case 3, mGSTP1 levels
the time of MRI testing, there was no HCC recurrence returned to not detectable in urine while TP53m was
detected from the visit. elevated in the urine collected 3 months later with
the MRI report of a 1.6-cm lesion. We speculate that
DISCUSSION the rising of the TP53 mutated clone was different
from the previously treated tumor nodule and was
This study demonstrates the potential applicability of either not responding to the treatment or was derived
using urine DNA markers in combination with serum from tumor evolution. Furthermore, it is possible that
AFP for the early detection of HCC recurrence in a apoptosis of a small tumor nodule through immune
small 10-case study. HCC recurrence is known to be system targeting could lead to a temporary rise in DNA
the major factor for poor prognosis. In this small 10- markers associating with that tumor. For example, for
case study, MRI identified recurrence in 5 out of 10 case 6 the mRASSF1A was found elevated at 3 years
patients (cases 1-5). Encouragingly, for all 4 recurrent and negative at 4 years post TACE, where recurrence
patients that remain in the study (cases 1-4), urine DNA was not detected.
markers were found to be elevated in urine samples as
early as 9 months before MRI confirmation. It is important to note that the levels of urine DNA
markers can fluctuate for several reasons including
Although this is a small longitudinal 10 patient hydration of the patient at time of collection (which
study, the potential of these urine DNA markers for can result in diluted DNA in the urine). Therefore,
management of HCC recurrence and important the use of an internal control is important for
characteristics of HCC recurrence is demonstrated. appropriately setting cutoffs for the urine marker
First, for all remaining recurrent cases (cases 1-4), values. While urine protein creatinine is the most
DNA markers were elevated before or at the time of used internal control for urine concentration, our
diagnosis by MRI imaging. MRI/CT imaging is the pilot study has suggested the concentration of LMW
gold standard for diagnosis of recurrent HCC, but has cell-free DNA in urine does not correlate with urine
difficulty in detecting early recurrence in the previously creatinine. Further studies are needed to identify
treated areas (especially after local ablation). This a proper internal control for this work, which is
may explain why the DNA markers were found in urine currently in progress.
earlier than MRI diagnosis. Secondly, HCC, like other
cancers, is a disease of the genome. Detection of In conclusion, we have demonstrated that urine DNA
genetic drivers of HCC may provide not only sensitive biomarker testing may have potential for the early
and earlier detection for monitoring HCC recurrence, detection of HCC recurrence. A larger longitudinal
but may also provide HCC genetic information to study design to collect well-annotated serial patient
assist in patient management. Furthermore, since samples is in progress, specifically to monitor for
collection of urine can potentially be done at home HCC recurrence, to test whether this urine DNA test
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