Review for 10.1186/s12876-021-01741-5 (2021)

Title: Evaluation of Circulating Cell-Free DNA in Cholestatic Liver Disease Using Liver-
Specific Methylation Markers
Version: 0 Date: 11 Feb 2021
Reviewer:  Roman Liebe
Reviewer's report:
Summary:
The authors' aim was to determine if liver-specific DNA was a good serum biomarker or
monitoring tool for PBC and/or PSC.
They identified 16CpG sites near 3 genes that were specifically unmethylated in liver tissue
while methylated in all other tissues.
Subsequently, they isolated cfDNA from peripheral blood and analysed the relative contribution
of unmethylated vs. total DNA by quantitative NGS of total PCR products following bisulfite
treatment.
The results show that unmethylated, i.e. liver-specific product concentration was higher in PBC
and PSC patients than in healthy controls, and higher in late-stage than in early-stage patients.
cfDNA levels were correlated with ALP, a serum marker used to characterise both diseases
investigated.
Hence, unmethylated products from these loci represent feasible markers of cell death, i.e. liver
damage in patients with cholestatic liver damage.
Questions:
- Methylation patterns for all CpG sites were quantified in total liver tissue, i.e. a gemisch
of cells? Is anything known about cell type specificity, i.e. hepatocytes vs. cholangiocytes vs.
epithelial cells?
- The quantification of unmethylated PCR products measures how much DNA from damaged
liver cells, i.e. apoptotic cells, is released into the blood stream.
That's a great way to assess hepatocyte damage, but the obvious questions are:
Is it specific for cholestatic damage, as ALP is? Obviously not, as the authors stress in the
introduction (ref #12).
What additional value beyond standard biochemistry is offered by the new approach?
Conclusions:
The central question is if the quantification of general liver damage from cfDNA in serum
samples adds any additional information beyond standard liver biochemistry (AST, ALT, GGT,
ALP).
The answer is "probably not", at least where fresh blood and routine biochemistry is available,
which provides a more granular view of liver damage by differentiating between several
enzymes from several sources, and the respective ratios (e.g. Ritis quotient)
As the authors state in the discussion, " the use of cholangiocyte-specific epigenetic marks may
prove more beneficial, particularly for PBC, in which cholangiocyte apoptosis plays a pivotal
role in pathogenesis".
The technical issues described in the discussion, i.e. sample collection and a complex technology
required for NGS quantification of differentially methylated sites, as well as a high level of
individual variation between results irrespective of disease state, pose serious questions as to
the usefulness in routine clinical diagnosis.
The experiments described in the manuscript are technically sound, provide sufficient detail,
and were analysed to the best extent.
The conclusion that a measurement of liver cell death can be derived from peripheral blood and
can be used to monitor progressive hepatic damage is sound, possible caveats outlined in the
discussion. PBC and PSC are two exemplary aetiologies that have been tested. Comparisons
with other aetiologies to provide any clue to specificity are missing.
The final statement that " cfDNA (1) offers promise to become a non-invasive liquid-biopsy to
evaluate liver-specific cell-death (2) in patients with cholestatic and other liver diseases" sums
up the promise (1) as well as the problem (2).