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New hepatitis B virus (HBV) therapies are expected to have breakthrough benefit for patients. HBV functional cure is sustained hepatitis B surface antigen loss and anti-HBs gain, with normalization of serum aminotransferases off therapy. Virologic or complete cure additionally includes loss of HBV covalently closed circular DNA. Currently available endpoints of therapy are inadequate to evaluate the efficacy of many of the new therapeutics. Therefore, either new ways of using the existing virologic endpoints and laboratory values or entirely new biomarkers are needed. In this review, we discuss the currently used endpoints, potential new endpoints, as well as what new markers are needed to assess the ability of HBV therapeutics to achieve functional and virologic cure in various phases of HBV infection. In addition, we discuss how patient selection from differing phases of HBV impacts the choice of HBV drug(s) needed to achieve cure., (© The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

The discovery in 1965 of the “Australia antigen,” subsequently identified as the hepatitis B virus surface antigen (HBsAg), was such a watershed event in virology that it is often thought to mark the beginning of hepatitis research, but it is more accurately seen as a critical breakthrough in a long effort to understand the pathogenesis of infectious hepatitis. A century earlier, Virchow provided an authoritative explanation of “catarrhal jaundice,” which did not consider an infectious etiology, but the transmission of jaundice by human serum was clearly identified in two outbreaks in 1885, and the distinction between “infectious” and “serum” hepatitis was recognized by the early 1920s. The inability to culture a virus or reproduce either syndrome in laboratory animals led to numerous studies in human volunteers; by the end of World War II, it was known that the diseases were caused by different filterable agents, and the terms “hepatitis A” and “B” were introduced in 1947 (though some long-incubation cases then designated B must in retrospect have been hepatitis C). The development of a number of liver function tests during the 1950s led to the recognition of anicteric infections and the existence of chronic carriers, but little more could be done until an infectious agent had been identified. Once Blumberg and colleagues had found a specific viral marker, the vast amount of accumulated epidemiologic and clinical data, together with huge numbers of stored serum samples, enabled rapid progress in understanding hepatitis B, and revealed the existence of a vast population of chronically infected people in Asia, Oceania and Africa. In this article, we place the identification of the Australia antigen within the historical context of research on viral hepatitis. Following a chronological review from 1865 to 1965, we summarize how the discovery led to improved safety of blood transfusion, the development of a highly effective vaccine and the eventual identification of the hepatitis C, D and E viruses. This article forms part of a symposium in Antiviral Research on “An unfinished story: from the discovery of the Australia antigen to the development of new curative therapies for chronic hepatitis B.” [ABSTRACT FROM AUTHOR]

Aim: Aberrant methylation of the promoter, P2, and the first exon, E1, regions of the tumor suppressor gene RASSF1A, have been associated with hepatocellular carcinoma (HCC), albeit with poor specificity. This study analyzed the methylation profiles of P1, P2 and E1 regions of the gene to identify the region of which methylation most specifically corresponds to HCC and to evaluate the potential of this methylated region as a biomarker in urine for HCC screening. Methods: Bisulfite DNA sequencing and quantitative methylation-specific polymerase chain reaction assays were performed to compare methylation of the 56 CpG sites in regions P1, P2 and E1 in DNA isolated from normal, hepatitic, cirrhotic, adjacent non-HCC, and HCC liver tissue and urine samples for the characterization of hypermethylation of the RASSF1A gene as a biomarker for HCC screening. Results: In tissue, comparing HCC (n = 120) with cirrhosis and hepatitis together (n = 70), methylation of P1 had an area under the receiver operating characteristics curve (AUROC) of 0.90, whereas methylation of E1 and P2 had AUROC of 0.84 and 0.72, respectively. At 90% sensitivity, specificity for P1 methylation was 72.9% versus 38.6% for E1 and 27.1% for P2. Methylated P1 DNA was detected in urine in association with cirrhosis and HCC. It had a sensitivity of 81.8% for α-fetoprotein negative HCC. Conclusion: Among the three regions analyzed, methylation of P1 is the most specific for HCC and holds great promise as a DNA marker in urine for screening of cirrhosis and HCC. [ABSTRACT FROM AUTHOR]

The Golgi phosphoprotein GP73 is elevated in the circulation of individuals with a diagnosis of hepatocellular carcinoma. Its usefulness as a biomarker of HCC is questioned, since it has also been reported to be elevated in the circulation of people with liver cirrhosis. Regulation of GP73 by inflammatory cytokines is therefore of interest. The interleukin-6 (IL-6) family cytokines were tested for effects on GP73 mRNA and/or protein levels in human hepatoblastoma HepG2 cells. Levels of GP73 mRNA and protein were up-regulated in HepG2 cells following treatment with either proinflammatory cytokine IL-6 or the related cytokine oncostatin M (OSM). Induction required the shared receptor subunit gp130, and correlated with increased tyrosine phosphorylation of STAT3. Maximal cytokine-mediated induction was not observed in the presence of protein synthesis inhibitor cycloheximide, suggesting additional regulatory factors play an important role. ELISA measurement of GP73 and IL-6 levels in the sera of patients with pre-malignant liver disease revealed a significant correlation between circulating levels of the two proteins. Similarly, a sensitive ELISA assay was developed to measure circulating OSM. OSM levels were elevated 6-7 fold in sera from patients with either cirrhosis or HCC relative to controls without liver disease. Although there was an association between levels of GP73 and OSM in serum from people with liver cirrhosis, there was not a statistically significant correlation in HCC, suggesting that the role of the cytokines in determining circulating levels may be complex. To our knowledge, this is the first report of OSM elevation being associated with liver disease. [ABSTRACT FROM AUTHOR]

We have previously demonstrated that mutated DNA derived from the circulation can be detected in urine and predominantly exists as DNA fragments <1 kb. To preferentially isolate the trans-renal DNA from urine, we developed a method using carboxylated beads to separate high-MW (1 kb or larger) from low-MW DNA in urine. A primer set for 18s rRNA (generating a PCR product of 872 bp) was designed and optimized for real-time PCR quantification of high-MW DNA templates. To evaluate the method, urine samples from 5 volunteers with no known diseases and 36 patients with various colorectal diseases were collected and tested. It was found that the average removal efficiency of high-MW DNA from total urine DNA using carboxylated beads is 92.72%± 1.42%. Furthermore, compared with using total urine DNA, our method provides a greater ability to detect mutated K-ras in the urine of colorectal cancer patients. The concurrence of K-ras mutations detected in disease tissue and the corresponding urine specimen is significantly higher ( P= 0.0015) when the samples were enriched in low-MW DNA. [ABSTRACT FROM AUTHOR]

Our previous studies demonstrated that urine contains DNA derived from the circulation and that this DNA originated, in part, from organ sites and tumors distal to the urinary tract. To explore the potential use of DNA from urine as compared to other body fluids as a source for circulating DNA for cancer detection, the DNA concentration and the frequency of detection of mutated Kristin- ras (K -ras) DNA in serum, plasma, and urine were examined. The concentration of DNA in the urine was similar to that in the serum, but the DNA concentration in plasma was significantly lower than in either urine or serum ( P < 0.05). When DNA derived from 10 μL of body fluid was used in each mutation assay, the detection frequency of mutated K -ras DNA was comparable among serum, plasma, and urine. However, when DNA derived from 200 μL of body fluid was used, the incidence of detecting mutated K -ras DNA in urine was significant higher (95%) than in either serum (35%) or plasma (40%) ( P < 0.0005), suggesting that inhibitory factors in serum/plasma may be more limiting than in urine. The use and practicality of urine as a source of circulating DNA for cancer detection are discussed. [ABSTRACT FROM AUTHOR]

This opinion piece evaluates the degree of readiness of 13 candidate biomarkers or panels of biomarkers for the early detection of hepatocellular carcinoma (HCC), or for the staging of liver fibrosis. As candidate biomarkers for the early detection of disease are identified, it is important to understand where they are in the developmental pipeline and how they might be employed. HCC is a growing public health problem, and its early detection is believed to be important in its management. Current detection methods are limited in usefulness or practicality, and there is a need for better methods to diagnose liver disease. Therefore, a number of candidate biomarkers, considered to be attractive because of their advanced stage of development or inherent scientific value, were evaluated for specificity and sensitivity for detection of liver disease. Study design, confirmatory evidence and assay practicality associated with each of the biomarkers are considered. The comments in this review reflect the authors' opinions and are based on a recent workshop convened by the Hepatitis B Foundation of America and the US National Cancer Institute's Early Detection Research Network. It is emphasized that only a selected set of biomarkers was considered; thus, this review is not comprehensive and not intended to review all candidate HCC biomarkers. [ABSTRACT FROM AUTHOR]

We previously demonstrated that human urine contains small, 150 to 250 nucleotide-sized, soluble DNA derived from the circulation, which may be useful in the detection of colorectal cancer. In this report we have determined the stability of DNA in urine and have found that the half-life time interval of this small, fragmented DNA is at least 4 hours post collection. We further compared, in a blinded study, the frequency of detecting mutated K-ras sequence in DNA isolated from plasma and urine derived from individuals who have either a colorectal carcinoma (CRC), or adenomatous polyps that contain a mutation in codon 12 of the K-ras proto-oncogene. There was an 83% concurrence of mutated DNA detected in urine and its corresponding disease tissue from the same individuals, when paired urine and tissue sections from 20 patients with either CRC or adenomatous polyps were analyzed for K-ras mutation. However, only a 56% concurrence was observed when the matched plasma specimens were tested from these 20 patients. These results suggest that urine might be a better resource for detecting K-ras mutation in circulating DNA. [ABSTRACT FROM AUTHOR]

A small portion of DNA from apoptotic cells escapes complete degradation, appears in blood as oligonucleosomal-size fragments, is excreted in the urine, and can be used for diagnostic purposes. More detailed study revealed that transrenal DNA (Tr-DNA) belongs to a relatively low molecularweight (150-250 bp) fraction, thereby requiring more careful attention to methods employed for purification and analysis. For example, here it is demonstrated that the QIAamp blood kit purifies primarily high molecular-weight DNA from serum, whereas the Guanidine/Promega Wizard Resin (GITC/WR) method purifies primarily low molecular-weight DNA. As a result, sensitivity in detection of K-RAS mutations in serum of patients with colorectal tumors is significantly higher with DNA isolated with the GITC/WR method than with the QIAamp kit. Amplicon size is also extremely important in analysis of Tr-DNA, because the shorter the amplicon, the higher is the sensitivity of biomarker detection in Tr-DNA, One hundred fifty-seven and 87 bp amplicons were employed for detection of mutant K-RAS in DNA isolated from 0.1 mL of urine obtained from 15 patients with pancreatic cancer. Mutant K-RAS was found in Tr-DNA of 3 and 10 patients with the long and short amplicons, respectively. The sensitivity and specificity of detection of mutant sequences are reduced in the presence of high excess of a respective wild-type allele, but they can be significantly increased through application of enriched polymerase chain reaction (PCR), peptide nucleic acid (PNA) clamped PCR, and/or stencil-aided mutation analysis (SAMA), based on selective pre-PCR elimination of wild-type sequences. [ABSTRACT FROM AUTHOR]