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Experts Call for Continued Focus on Genetics, Genomics in DAA Era

In an era when ‘special’ populations within hepatitis C — even those with resistance-associated variants — begin to shrink out of sight, questions arise about the utility or necessity of comprehensive genomic testing and research in HCV. Most experts believe that knowing a patient’s genotype remains a critical piece of information, along with the presence or absence of baseline RAVs. But whether the research community should continue moving forward with exhaustive analysis of other genomic information about the virus could be up for debate.

For Anita Howe, PhD, scientific leader for HCV at the British Columbia Center for Excellence for HIV/AIDS, the fundamental issue is potential development of drug resistance, or multi-drug resistance.

“Now that we are getting over 90% [sustained virologic response], people have become a little bit short-sighted,” she said in an interview with HCV Next. “They think there is no need to put investment into drug discovery. This brings us back to the resistance mutation situation. Even now, with current DAAs, if you fail first-line drugs, the same mutation is likely to cause resistance to second-line drugs. How are we going to deal with these resistant patients? We are trying to alert the community, but we sound like a broken record.”

David L. Wyles, MD, associate professor of medicine at University of California, San Diego, School of Medicine, acknowledged the reality of resistance but recognized the few this would affect.

“First off, there is a very small portion of non-responders,” he said. “I agree that we still don’t have well-defined salvage therapies for those patients. Currently, the standard practice would be to get RAV testing. But there is still not concrete evidence that adjusting therapy to those RAVs would be beneficial.”

A logical solution, for Wyles, is to wait for the next round of therapies to hit the market. “They will be triple or dual therapies, with pangenotypic activity, higher barriers to resistance, and activity against some common resistant variants,” he said. “They will be one-size-fits-all — well, at least most. RAV testing may still be used to let us know if we need to add ribavirin or cannot use short duration therapy.”

Recent approval of the co-formulated, pangenotypic sofosbuvir/velpatasvir (Epclusa, Gilead) may portend a future when such therapies become first-line therapies. But the issues of genomics in HCV run deeper than predicting therapeutic response.

HCV Next addressed topics ranging from microRNA-122 to alleles and polymorphisms that continue to undergo investigation despite so many research dollars aimed at DAAs. Experts weighed in on genetic predictors of cirrhosis and HCC development along with the role of genomic information in finding an HCV vaccine. But it is the necessity of finding therapies that prevent drug resistance that may bring genomic studies back to the forefront.

Oncoming Resistance

Howe said most pharmaceutical companies discontinued new drug discovery programs for HCV. “It is up to us in the community — and that includes physicians and researchers alike, along with pharma — to continue to do resistance testing,” she said. “Let us not forget that even with SVR rates over 90%, the remaining 5% to 10% could have multidrug resistance. We don’t want to repeat a similar situation with antibiotics, where we thought we had gotten rid of bacterial infections, but then they came back stronger.”

She warned against complacency in the clinical community. “The success of these drugs has caused us to turn our attention elsewhere at a critical moment,” she said.

Fortunately, Howe and others continue to ring the warning bell. “We are setting up a resistance database,” she said. “It’s a start.”

It is largely a numbers game, according to Howe. “There are 180 million people with HCV worldwide,” she said. “Even if you have 5% failure, this is still a huge number of people. If you have no drugs for those people, they will just keep transmitting.”

Howe acknowledged that most baby boomers, even those who are not treated or who are treatment failures, are not transmitting the disease. “The other major population, which includes young people using needles, [men who have sex with men] and commercial sex workers, is a major concern,” she said. “The transmission rates in these populations are significantly higher than in baby boomers. Even at a 5% failure rate, if these core transmitters continue to spread the disease, this will be a major issue.”

Alex Thompson, PhD, MBBS, director of gastroenterology at St. Vincent’s Hospital in Melbourne, Australia, offered a counterpoint: “Sequencing of the virus remains relevant, particularly with respect to NS5A variants and drug resistance.”

Wyles agreed. “Clinicians will be genotyping HCV for some time, for a number of reasons,” he said. “One is that genotyping information is valuable to us. For example, we know that genotype 3 can lead to a rapid progression to steatosis. Or if grazoprevir and elbasvir are the primary treatment regimen, you have to do NS5A RAV testing.”

Also, payers likely will continue to require genotyping information for coverage, according to Wyles. “Genotyping information is still dictating treatment in a lot of ways,” he said. “There is still utility in knowing the genotype and doing RAV testing, and this information will benefit us in the future.”

Anthony J. Michaels, MD, of the departments of gastroenterology and hepatology at the Ohio State University Wexner Medical Center, built on this: “This information can help us learn which patients need extended therapy and which patients need ribavirin. ... We are moving toward individualized therapy, rather than a shotgun approach, and genetic and genomic information can help us make those determinations.”

Progression to Cirrhosis, HCC

A further argument for conducting more comprehensive testing of genetic information in HCV is the association between certain markers and progression to cirrhosis and hepatocellular carcinoma. Moreira and colleagues assessed the genotype of 22 SNPs found in the genes of 13 cytokines or cytokine receptors to determine if they influence polymorphic variants in the stage of liver damage in a cohort of patients with HCV infection genotype 1. They reported an association between the TNFA-308G:A genotype and increased fibrosis and cirrhosis risk. Conversely, the TNFA-238G:G genotype conferred a protective effect against cirrhosis. The IL10-819C:T genotype had a protective effect against fibrosis, while the IL1B-511C:T genotype yielded an increased cirrhosis risk. “We conclude that gene variants of cytokines/receptors may influence liver damage in patients chronically infected by HCV genotype 1,” the researchers wrote.

“What we are seeing that is causing increasing concern is a persistent risk of HCC even if HCV is eradicated,” Wyles said. “There are true genetic markers that can be found that reliably predict risk for HCC. These can be very useful.”

Untangling the knots of genetic information, therapeutic response and post-treatment care is part of the challenge, according to Wyles. “What we are struggling with right now is how long do we need to screen patients for HCC after they are cured,” he said. “Now, we are doing it indefinitely. If there was a genetic marker to predict high-risk populations, then we can figure out who to screen or come up with a shorter screening duration post-SVR.”

In another study, Ulveling and colleagues conducted a genome-wide association study to determine factors associated with liver fibrosis progression in a cohort of patients coinfected with HIV and HCV. They obtained two signals of genome-wide significance (P < 5 x 10-8). The first was on chromosome 3p25 and corresponding to rs61183828 (P = 3.8 x 10-9). The researchers replicated this in two cohorts of patients monoinfected with HCV. “The cluster of SNPs in linkage disequilibrium with rs61183828 was located close to two genes involved in mechanisms affecting both cell signaling and cell structure (CAV3) or HCV replication (RAD18),” they wrote. They did not replicate the second signal, which they obtained with rs11790131 (P = 9.3 x 10-9) on chromosome region 9p22, according to the results. The researchers concluded that the findings provide a novel hypothesis for the pathogenesis of fibrosis that may aid in drug development.

“There is still much interest in the genetic determinants of liver fibrosis progression, as well as risk markers for HCC, to inform prognostic assessments as well as to identify novel pathogenic mechanisms that may inform therapeutic development,” Thompson said. “We are nowhere near this with any of these markers.”

What exists is a growing body of data that, for some experts, including Michaels, may become unwieldly. “There is so much information out there from these gene studies,” he said. “We don’t know what to do with it all.”

Wyles offered a practical suggestion: “These markers need to be studied prospectively, because we still struggle for markers of cirrhosis and HCC. Right now, the standard of care is to continue to screen indefinitely. The studies to determine these risks will need to be longer than 5 or 10 years. It is a pretty tall order, and we are quite a way off.”

miRNA122

One area that is understood and translating into a therapeutic option is miR-122. A landmark study presented by Meike van der Ree, MD, at the International Liver Congress 2015 showed a single injection of RG-101 (Regulus), an oligonucleotide with activity against miR-122, yielded undetectable viral load patients of multiple genotypes. Patients received either administration of 2 mg/kg RG-101, 4 mg/kg or placebo. Eight-week results indicated 54% had HCV RNA levels below the limit of quantification. The researchers extended the follow-up period and interim results were reported for 20 weeks. At that time, 70% of patients still being followed remained below the limit of quantification. The drug was well tolerated.

Howe put the findings into context. “These data are quite promising,” she said. “The half-life of this injection is very long. You can afford to have a monthly injection.”

Perhaps more importantly, the resistance profile of the treatment is attractive, according to Wyles. “Since you are interrupting a host miRNA process, there is less chance of developing resistance,” he said. “The sequence of the 5’-UTR also determines the structure of the RNA, placing additional constraints viable variants which creates a high barrier to resistance.”

An important drawback of the injection approach, according to Howe, is that many of today’s HCV patients, particularly patients in the developed world, are accustomed to oral therapies. “An injection could be logistically difficult,” she said. “Compliance and delivery need to be worked out, and more importantly we need to monitor the safety profile.”

In June, the FDA placed a clinical hold on RG-101 due to jaundice in a patient with end-stage renal disease on dialysis 117 days after receiving a single dose of the medication. This hold will not affect ongoing trials.

At the International Liver Congress in 2016, Gabor Horvath, MD, presented data on combining RG-101 with 4 weeks of DAA treatment.

In this interim report, HCV RNA levels were below the lower limit of quantification at week 12 post-dosing in 100% of patients treated with RG-101 and ledipasvir/sofosbuvir (Harvoni, Gilead Sciences), 93.3% treated with RG-101 and simeprevir (Olysio, Janssen) and 100% treated with RG-101 and daclatasvir (Daklinza, Bristol-Myers Squibb).

“When we can figure out how to give the injection and the orals together, we could give [the injection] relatively infrequently. This could lead to an ultra-short duration with a high barrier of resistance that may carry advantages over all oral therapies,” Wyles said.

For Michaels, there can never be enough medications that directly attack the virus. “This injection directly attacks the virus at the viral life cycle,” he said. “It is pretty amazing that one shot achieved this.”

Alleles, Polymorphisms

Despite reservations about the overwhelming body of genomic information, research is ongoing. Hueging and colleagues studied Abhydrolase Domain-Containing Protein 5 (ABHD5). They suggested that HCV replication is associated with the way lipids are metabolized by the host and suggested that ABHD5 could be a host factor for the assembly and release of the virus.

The findings indicated that a transfer of lipids that is dependent on ABHD5 is required for the maturation of very low density lipoprotein (VLDL) coupled with the assembly of HCV and virion release. In turn, then, lipid remodeling may affect assembly and virus transport, according to the results.

The researchers added that ABHD5 is associated with Chanarin-Dorfman. “We found that the Chanarin-Dorfman syndrome mutants were unable to support HCV assembly, pointing at a new host polymorphism that could determine susceptibility to HCV infection,” they wrote.

In another study, Jung and colleagues suggested the role of apolipoprotein E in HCV replication remains unclear. They aimed to elucidate the role of this protein in the replication process in genotypes 1b and 2a. Results indicated that viral suppression was achieved by knocking down expression of apoE with a specific RNA that interfered. This suggests the necessity of this protein in infectious HCV particles, according to the researchers. Replication of the virus was not impacted in cells treated with each isoform of the protein or transfected with apoE–specific siRNAs while knocking down the expression of the protein suppressed replication in patients in the genotype 1b group. Additionally, the siRNA-mediated knockdown of apoE, apoA1 and apoB expression also resulted in suppression for genotype 1b, but not for 2a.

“These findings indicate that apoE plays an important role in HCV genotype 2a infection and in HCV genotype 1b RNA replication, but not in the replication of HCV genotype 2a,” the researchers concluded. “These results provide important information for the future development of HCV-genotype specific anti-HCV agents.”

Michaels acknowledged that these studies can provide information on the natural history of the virus. “Some of these data on the evolution of the viral quasispecies may someday impact treatment,” he said. “The fact remains, though, that we are still getting very high rates of cure.”

Finding, Using Genetic Information

Emma Thomson, PhD, FRCP, and colleagues investigated next generation sequencing technologies that could identify genotype and resistance information for DAA therapies. They looked at metagenomics methods using unselected HCV RNA, pre-enrichment of HCV RNA by probe capture and pre-amplification of the disease by PCR, according to the results. Using viral load data, along with genotype, they evaluated metric of sequence coverage and depth, quasispecies diversity and detection of RAVs, in addition to information about co-infections and mixed genotype HCV. All of the approaches were able to generate almost complete genome sequences for the majority of samples, and all were able to identify mixed genotype disease.

Greater sequence depth was generated by enrichment methods and PC pre-amplification, according to the results. These approaches also were more useful for samples with low viral loads, while enrichment methods are recommended for high-throughput analysis to offer genotype and resistance information. Metagenomic approaches were more likely to identify pegivirus coinfections.

“Consensus sequences generated by different [next generation sequencing] methods were generally concordant and majority RAVs were consistently detected,” they wrote. “However, methods differed in their ability to detect minor populations of RAVs.”

“These assays allow us to sequence the whole genome, which, in turn, can help us target genes,” Howe said. To her knowledge, the BC Centre for Excellence in HIV/AIDS is the only laboratory in Canada that provides whole genome next generation sequencing to patients with HCV.

But most places around the world do not have the resources to have entire labs dedicated to exploring genomic information in HCV. Despite the obstacles, Thompson acknowledged the importance of genomic testing in the big picture. “It should be possible to eradicate HCV in the developed world in the next 10-plus years,” he said.

Some have advocated the use of genomic information in developing a vaccine, but this effort has not moved forward. “There have been efforts to make a vaccine in the past,” Michaels said. “It has not worked very well.”

Another inactive area is investigation of the IL28B polymorphism. “IL28B is no longer recommended as a routine test before DAA therapy,” said Thompson, who has studied the polymorphism extensively. “It remains a research tool, and may be relevant to very short duration DAA regimens, where patients who carry the ‘good responder’ genotype may be eligible for shorter duration regimens.”

Wyles agreed, but offered a qualification. “This variant helped us understand who would respond to interferon,” he said. “It hasn’t played a significant role in DAAs. But it may still have use in places where interferon is the only option.”

In the end, then, it comes back to ensuring the therapies on the market are being used with optimal efficacy and efficiency. “We need to determine who can achieve SVR in a shorter duration or with less therapy,” Michaels said. “Knowing what patients have these RAVs, and therefore need a longer duration or ribavirin, can be useful.”

Howe added that demographic information can work in concert with the genetic and genomic information. “We should pay attention to certain populations such as patients with cirrhosis or high BMI,” she said. “People with advanced liver disease and perhaps African American men may be more susceptible to the impact of baseline mutations than other populations.

There is no doubt that exploratory analysis in HCV genomics is decreasing, according to Wyles. “It may continue to some extent, but attention is elsewhere,” he said. “I hope that it won’t matter terribly as long as patients are adhering to current and next generation therapies.”

• References:
• Gomaa HE, et al. Open Access Maced J Med Sci. 2015;doi:10.3889/oamjms.2015.046.
• Horvath G. Abstract GS08. Presented at: International Liver Congress; April 13-17, 2016; Barcelona.
• Hueging K, et al. J Virol. 2014;doi:10.1128/JVI.01815-13.
• Jung BK, et al. J Microbiol. 2016;doi:10.1007/s12275-016-6099-3.
• Moreira ST, et al. Meta Gene. 2016;doi:10.1016/j.mgene.2016.04.003.
• Thomson E, et al. J Clin Microbiol. 2016; pii: JCM.00330-16. [Epub ahead of print].
• Ulveling D, et al. Hepatology. 2016;doi:10.1002/hep.28695.
• van der Ree M. Abstract L07. Presented at: International Liver Congress; April 22-26, 2015; Vienna.

• For more information:
• Anita Howe, PhD, can be reached at BC Centre for Excellence in HIV/AIDS, St. Paul’s Hospital, 608–1081 Burrard Street, Vancouver, BC, Canada V6Z 1Y6; email: dpepin@cfenet.ubc.ca.
• Anthony J. Michaels, MD, can be reached at 410 W 10th Ave, Columbus, OH 43210; email: Marti.Leitch@osumc.edu.
• Alex Thompson, PhD, MBBS, can be reached at Department of Gastroenterology, SVHM, Level 4 Daly Wing, 35 Victoria Pde, PO Box 2900, Fitzroy, Victoria 3065, Australia; email: alexander.thompson@svha.org.au.
• David L. Wyles, MD, can be reached at UCSD, 9500 Gilman Drive, Mail Code 0711, La Jolla, CA 92093-0711; email: dwyles@ucsd.edu.

Disclosures: Howe reports no relevant financial disclosures. Michaels reports being a speaker for Gilead and being on the advisory boards of Bristol-Myers Squibb and Gilead. Thompson reports acting as an advisory board member for AbbVie, Bristol-Myers Squibb, Gilead Sciences, Merck and Roche; receiving research and grant support from AbbVie, Alnylam, Arrowhead, Bristol-Myers Squibb, Gilead Sciences, Merck and Sillajen; and acting as an investigator for Alnylam, Arrowhead, Bristol-Myers Squibb, Gilead, Merck and Spring Bank. Wyles reports receiving research grants for the conduct of clinical trials paid to UC Regents from AbbVie, Bristol-Myers Squibb, Gilead Sciences, Merck and Vertex, and serving as a paid consultant to AbbVie, Bristol-Myers Squibb, Gilead Sciences and Janssen.