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Ding et al. Hepatoma Res 2019;5:10 I http://dx.doi.org/10.20517/2394-5079.2018.115 Page 5 of 8

Although these studies mentioned above showed that serum HBV RNA may have great potential to act as a
supplementary biomarker for judging the effect of antiviral therapy, it remains unclear whether serum HBV
RNA is superior to existing biomarkers, or whether it can replace other biomarkers for the same clinical
applications.

Monitoring safe discontinuation of NA-therapy in CHB patients
As HBV cccDNA cannot be completely cleared during NAs therapy, most CHB patients have been suffering
from virological rebound and HBV relapse, making it difficult to decide the timing of NAs therapy
withdrawal. So, the majority of CHB patients have to receive NAs therapy for a long time, even their entire
lifetime, which aggravates the financial burden for both the patients and the society [26,27] . As mentioned
above, serum HBV RNA could be regarded as a potential indicator for cccDNA activity, so the vanishment
of serum HBV RNA may represent the transcription silence of cccDNA. Therefore, serum HBV RNA could
serve as a potential predictable marker for safe withdrawal of NAs therapy [24,32] . A study on 36 CHB patients
treated with NAs for at least 6 months revealed that after discontinuation of NA therapy for 24 weeks,
their HBV DNA and HBV RNA titer on the third month of treatment was significantly associated with
[33]
HBV DNA rebound and alanine aminotransferase rebound . Another study of 33 CHB patients who had
received NAs treatment for at least 3 years and whose serum HBV DNA was undetectable afterwards showed
that all patients with HBV RNA positive experienced virological rebound at the end of treatment after
withdrawal of NAs for 24 weeks, while virological rebound occurred in only 25% of patients with negative
[8]
serum HBV RNA . However, as the sample size of these studies and the follow-up time are insufficient,
additional studies with larger sample size and longer follow-up time are needed to further verify whether
HBV RNA can be used as a predictive biomarker to reflect the rebound of HBV after discontinuation of
antiviral treatment.

To assess the prognosis of HBV-associated HCC
Few studies have reported the relationship between HBV RNA and the prognosis of HBV-associated HCC.
A recent study on 99 HBsAg-positive, virologically suppressed patients treated by tumour resection or liver
transplantation indicated that HBV pgRNA was detectable more frequently in non-tumor (55/61; 90%)
than in tumor samples (40/60 (67%); P < 0.01). When detectable in both compartments, the levels of pgRNA
were slightly higher in non-tumor than in tumor samples. Moreover, the detection of pgRNA in HCC is
significantly associated with lower incidence of vascular invasion and better survival rate. HCC expressing
higher HBV pgRNA may represent a kind of well differentiated, less-proliferative and low-invasive HCC
[7]
subtype . Therefore, HBV pgRNA might be used as a new biomarker for assessing the prognosis of HCC.
However, in consideration of the insufficient sample size of this study, further research is still needed.
Moreover, serum circulating HBV RNA may act as a biomarker for predicting the occurrence of HCC,
which needs further study [21,22] .

THE MEASUREMENT OF HBV RNA
[34]
For the first time, Kock et al. successfully detected the HBV RNA in the serum of CHB patients through
the method of rapid amplification of complementary DNA (cDNA)-ends (RACE) in 1996.The specific
primer with a special anchored sequence was used to form cDNA after the extraction of HBV RNA from
CHB patient serum. To ensure the high specificity for HBV RNA amplification, cDNA was amplified by
PCR with HBV-specific forward primer and the reverse primer which is identical to the special anchored
sequence. Since then, similar methods have been used to detect intrahepatic and serum HBV RNA in CHB
[37]
patients [35,36] . Using unique primers designed for reverse transcription, Kairat et al. developed RACE-
based real-time quantitative PCR to specifically quantify serum 3’ flRNA and 3’ internally truncated
polyadenylated HBV RNA later. Conventional RT-qPCR method with HBV-specific primers was also used to
quantify intrahepatic and serum HBV RNA. However, DNase I pretreatment of the nucleic acids extracted
[7,8]
is necessary to avoid DNA contamination before RT-qPCR . Recently, super-sensitive droplet digital PCR
[6]
was used to quantify serum HBV RNA by Wang et al. with HBV-specific primers. Collectively, many