Comprehensive Global Testing Solutions for HCV Novel

LABCORP’S STRENGTHS IN CLINICAL TRIALS AND
THE BENEFITS OF PATIENT TESTING APPLICATIONS
Comprehensive Global
Testing Solutions for HCV
Novel Therapeutics in
Clinical Development
remain unaware they are harboring the virus. This long
asymptomatic phase has earned HCV the nickname the
“silent epidemic,” making health authorities fear that
data on new infections might
vastly underestimate the scale of
the problem. If current estimates
of 3 million new infections per
year are accurate,6 many people
Christos J. Petropoulos, Ph.D.
Chief Scientific Officer, Monogram Biosciences, a LabCorp Company
Patrice Hugo, Ph.D.
Chief Scientist, LabCorp Clinical Trials
are destined to suffer from HCVrelated illnesses in the coming
decades if not given aggressive
and effective treatment
interventions.
HEPATITIS C VIRUS – BIOLOGY
AND CLINICAL IMPLICATIONS
Seventy-five percent of HCV
infections result in chronic
infection, and 60% of these cases develop severe
liver disease.7 Liver cancer and cirrhosis are among
The identification of the hepatitis C virus (HCV)
in 1989 solved a puzzling healthcare mystery.
Throughout the 1970s and 80s, doctors treated
patients presenting with hepatitis-like symptoms, but
the worst outcomes, resulting in 5% mortality in those
infected. In the United States, chronic HCV infection
is the leading indication for liver transplantation and
is the most common cause of liver disease-associated
mortality. Hepatocellular cancer is the fastest growing
cause of cancer-related mortality with approximately
were unable to assign causation to either hepatitis A
50% caused by HCV infection. More than 350,000
or B virus infection. The discovery of HCV pinpointed
people worldwide die from hepatitis C-related liver
the etiological agent of these cases, and a year later
doctors had a diagnostic test available to identify
infected individuals.¹
The ability to detect HCV enabled health officials to better capture
the global incidence of infection. The data showed HCV infection
was a widespread problem. In 1990, more than 2% of people
worldwide were infected with HCV.² By 1991, doctors were using
the immune modulator interferon (IFN) alpha to treat HCV, and in
the late 90s, IFN-alpha treatment was augmented with a second
therapeutic agent, ribavirin, that improved response rates.³ In 2005,
an estimated 180 million people worldwide were infected with
HCV. Current estimates now suggest that approximately 200 million
people are infected globally, of which approximately 170 million are
chronic carriers.4,5
Typically, 20 years might pass between acute HCV infection and the
disease every year. The problem is compounded by
inconsistencies in the efficacy of treatment. Drugs that
are highly effective in some patients have little effect in
others. In other cases, patients experience such severe
adverse side effects that they choose not initiate or
continue therapy.
Given the high proportion of the “baby boomer”
generation (individuals born between 1945-1965)
that is infected with HCV, the need for efficacious and
cost-effective treatments is now greater than ever. It is
estimated that the number of Americans with advanced
liver disease will soar as many HCV-infected subjects
enter their symptomatic phase of infection.8 In 2008,
an estimated 195,000 HCV-infected subjects suffered
from advanced liver disease, such as cirrhosis and liver
cancer. By 2015, this number is expected to approach
600,000.
onset of symptomatic liver disease, during which time many persons
labcorp.com/clinicaltrials
1
THE TROUBLESOME
GENETICS OF HCV
In 2011, a breakthrough in therapy came with the first two direct
acting anti-HCV drugs came to market and were followed by the
market approval of two additional anti-HCV drugs in 2013. While
these new drugs have significantly improved HCV cure rates,
the natural variation among HCV strains continues to challenge
treatment success across all populations of HCV infected patients.
HCV has 11 major genotypes,9 some of which respond better to
certain treatments than others, and treatment success can vary
dramatically based on the integrity of liver function (i.e. disease
stage). HCV is an enveloped RNA virus with a highly mutable
genome. The absence of RNA proofreading during viral replication
means that HCV is constantly mutating, which enables it to escape
from host immunologic detection and elimination. In large part, this
Developers of antiviral drugs benefit from insights on the
emergence of resistance and other obstacles to effective
treatment of HCV. Routine drug resistance assays provide
this knowledge. Resistance assays identify HCV-infected
patients who are more likely to respond to treatment
by identifying resistance-associated mutations that
may compromise antiretroviral regimens. In 1990, the
development of the first HCV diagnostic assay marked the
start of a 20-year struggle to combat HCV infection. Now,
drug resistance assays are poised to facilitate a new era of
antiviral HCV treatments.
is why so many people who are infected with HCV become chronic
cases, and also why researchers have so far failed to develop an
effective vaccine.10 The same high rate of mutability also helps HCV
develop resistance to antiviral drug treatment. Direct acting antiviral
agents targeting NS3/4A protease, the NS5A protein, and NS5B
polymerase are changing the paradigm in HCV treatment, but the
ability of HCV to rapidly develop resistance to these agents is a
APPLYING PATIENT
GENETICS TO
IMPROVED CARE
threat to their widespread, long-term efficacy. NS3/4A, NS5A and
NS5B are non-structural proteins involved in the replication of HCV.
The most recent era of HCV diagnostics was broadened
Inhibiting one or more of these proteins arrests virus replication, but
by a study published in Nature in 200911 by scientific
similar to HIV, incomplete suppression of viral replication by direct
teams from Merck & Co and Duke University, which
acting agents gives rise to resistant strains that are much more
described the importance of the IL28B genetic
difficult to treat or cure.
polymorphism. They found that patients with the
homozygous CC genotype for a particular single
HCV Direct Acting Agents Target Protease,
Polymerase and NS5A Activities
nucleotide polymorphism (SNP), named rs12979860,
have a stronger immune response to
HCV infection, allowing them to clear
HCV infection more frequently even
in the absence of treatment. The
homozygous CC genotype is also
associated with improved rates of
sustained viral suppression following
treatment with pegylated interferon
alpha-ribavirin therapy.
Soon after this publication, LabCorp
licensed the global rights to IL28B testing and began
Adapted from Kwong AD. Curr Opin Pharmacol 2008; 8(5); 522-31.
2
labcorp.com/clinicaltrials
offering the assay to assist in patient treatment decision
making and clinical trial enrollment. IL28B assays now
contribute to the more than 1 million HCV-related tests performed
at LabCorp each year. IL28B testing is performed at LabCorp’s
specialty testing laboratories, including Monogram Biosciences and
The Center for Molecular Biology and Pathology (CMBP). LabCorp
also offers IL28B testing for use in drug development and clinical
studies through the company’s clinical trials division, which has
supported more than 100 hepatitis-related clinical trials to-date.
In clinical trials, LabCorp’s IL28B and HCV genotype/subtype test
offerings help drug developers select suitable patients for study
enrollment, or prospectively and retrospectively characterize
baseline samples. Additional on-treatment monitoring assays,
such as quantitative viral load measurements provide insights
into whether treatment arms, or individuals within the group, are
responding to therapy. LabCorp’s comprehensive HCV portfolio
includes sensitive, state-of-the-art drug resistance assays for
characterizing treatment failures that include suboptimal viral
suppression or breakthrough while on treatment, or relapse after
completion of treatment.
LabCorp’s broad portfolio of HCV tests has applications from
preclinical services, through all phases of clinical development
and into post-marketing stages and commercial patient testing.
Testing is conducted at College of American Pathologists (CAP)accredited clinical reference laboratories staffed by well-trained
and experienced technologists with attention to quality assurance
and regulatory compliance. Testing is supported by technical
staff specializing in assay development,
automation and bioinformatics.
DEVELOPING THE NEXTGENERATION OF HCV TESTS
At LabCorp, externally-developed tests are used
alongside our in-house laboratory developed assays.
Having an experienced in-house assay development
team allows LabCorp to respond quickly to critical
It is the highly experienced assay
scientific advances and client requests, rather than
development staff, along with long-
relying on third party innovation. These in-house
standing and close ties to manufacturers of
capabilities are particularly important in rapidly
diagnostic kits, reagents, and devices that
evolving fields, like today’s HCV drug development
has positioned and maintains LabCorp at
efforts. LabCorp quickly appreciated that many drug
the forefront of HCV testing technology. Our
developers lacked tools to characterize the emergence
relationship with Merck enabled LabCorp to secure an exclusive
of drug-resistant variants during antiviral drug
license for the IL28B marker and typifies how the company can
treatment. In response, teams of LabCorp scientists
move quickly to add new, state-of-the-art, externally-developed
began developing tests to identify drug-resistance
tests. LabCorp was also one of the first laboratories to offer Abbott’s
associated mutations and to measure their effects on
RealTime HCV assay for measuring viral load. Furthermore, in June
drug susceptibility. Much of this work draws on the
2013 LabCorp became the first major clinical reference laboratory
experiences of Monogram Biosciences, gained through
to offer Roche’s more sensitive quantitative viral load assay, COBAS®
its pioneering development of high-throughput HIV
AmpliPrep/COBAS® TaqMan® HCV Test, v2.0. This sensitive assay is
drug-resistance assays.
now being offered for clinical trials globally, including in the LabCorp
facility in Beijing, China.
labcorp.com/clinicaltrials
3
HCV GenoSure Assays
LabCorp also offers cell-based infectivity assays to directly
assess reductions in susceptibility to NS3/4A, NS5A
and NS5B inhibitors. Such so called “phenotyping”
assays, including PhenoSense, are an essential tool
in establishing the relationship between particular
NS5B Amino Acid Differences from Reference
S5T, V11I, A117N, K212R, N444D, S506N, R531K, G543S, R544Q, H566R, L588Y
ID 0303
LabCorp has multiple HCV antiviral drug-resistance assays based
on DNA sequencing (GenoSure®) and cell-based infectivity
(PhenoSense®) platforms that are either fully validated or at
advanced stages of development. For prospective clinical studies
that require CLIA/CAP-compliant assays, such as those involving
a clinical management decision (e.g., as a patient selection
tool), LabCorp offers validated GenoSure assays that identify
mutations that confer resistance to NS3/4A protease, NS5A and
NS5B polymerase inhibitors. GenoSure assays are performed by
isolating viral RNA from a plasma sample and performing a reverse
transcription reaction to generate a cDNA copy that is subsequently
amplified by polymerase chain-reaction (PCR). NS3/4A, NS5A and
amino acid substitutions and quantitative reductions
in drug susceptibility. The correlations of “phenotype”
to “genotype” are particularly important during the
preclinical development and clinical evaluation of new
drugs and new drug targets. LabCorp’s PhenoSense
assays are conducted by incorporating patient derived
HCV sequences into an HCV luciferase reporter replicon,
which is then evaluated for replication in the presence
of increasing concentrations of drug. Data are typically
reported as the concentration of the drug required to
inhibit virus replication by 50% (IC50) or 95% (IC95),
relative to a reference virus strain. LabCorp clients have a
large degree of flexibility in specifying the data metrics and
formats that they prefer to receive from both genotypic
and phenotypic drug resistance tests.
NS5B amplification products serve as the template for conventional
LabCorp is also working on several initiatives to improve
Sanger sequencing reactions. Based on the constellation of
the sensitivity to detect minor drug resistant variants and
drug resistance mutations detected, LabCorp GenoSure reports
one approach has been the development of a proprietary
provide predictions of drug susceptibility that include “sensitive,”
measure of minor variant detection that is based on the
“resistance possible” and “resistant” interpretations.
slope of inhibition curves, which is significantly more
sensitive than conventional IC50 or IC95 measurements.12
PHENOSENSE HCV ASSAYS
Plasma sample
q
Virion lysis and viral RNA capture
q
Target sequence amplification (RT-PCR)
q
Transfer into luciferase reporter replicon
q
Linearize replicon DNA and in vitro transcribe vRNA
q
Electroporate cured Huh7 cells with vRNA
q
Measure luciferase activity at 4 and 72-96 hours (-/+ inhibitor)
q
Report susceptibility curves, IC50 & IC95 fold-changes from reference
Sample ID
10_XXXX
4
labcorp.com/clinicaltrials
IC50 Fold-Change from Reference (Con1)
Interferon Ribavirin Inhibitor A Inhibitor B Inhibitor C
1.2
0.9
1.3
9.5
32.4
TH E F U TU R E O F
H CV TESTI N G
Establishing the slope of the inhibition curve as an
additional accepted metric of drug resistance is one
of many innovations that LabCorp is implementing to
enhance the value of its HCV testing capabilities. LabCorp
is also aggressively pursuing HCV drug resistance assays
that can be performed on diverse HCV genotype/subtype
HCV RESISTANCE ASSAY APPROACHES
POPULATION ANALYSIS
Sequence and phenotype of patient virus populations
CLONAL ANALYSIS
Sequence and phenotype of individual molecular variants
VIRUS PANELS
HCV RESISTANCE ASSAY INITIATIVES
Reference replicons containing mutations that are associated with resistance
to HCV protease, polymerase or NS5A inhibitors (site‐directed mutants)
ENHANCED DRUG RESISTANCE ASSAYS
FOR NON‐GENOTYPE 1 VIRUSES
Replicons containing patient‐derived sequences that confer resistance
to HCV protease, polymerase, or NS5A inhibitors
Conventional sequencing (genotypic resistance assays)
NEXT GENERATION SEQUENCING
Parallel (deep) sequencing of viral quasi‐species
Replicon susceptibility (phenotypic resistance assays)
Non‐genotype 1 reporter replicons (e.g., JFH‐1)
NEXT GENERATION SEQUENCING ASSAYS
Platform evaluation
populations. Although the first generation of direct acting agents are
Consensus and minor variant determinations
limited to the treatment of HCV genotype 1 infections, which comprise
Specific drug targets (NS3/4A, NS5A, NS5B)
70% of hepatitis C patients in the United States,13 second generation drugs
Whole genome sequencing
with broader activity are currently undergoing intensive clinical evaluations.
ADDITIONAL DRUG TARGETS
NS4B, entry
In response to these efforts, LabCorp is actively working to expand
GenoSure and PhenoSense testing to HCV patients with non-genotype 1
viruses. Through these efforts, LabCorp is currently capable of conducting
genotyping and phenotyping assays for many HCV genotype 2a/2b, 3
and 4 strains and we are pursuing additional replicon systems to further
expand HCV genotype 2, 3 and 4 testing capabilities.
More aggressive screening and treatment programs
have been developed to reduce the health and financial
burdens of advanced HCV infection, and there are early
indications that these efforts are producing beneficial
results. In the past year the United States Centers for
Disease Control and Prevention (CDC) and United States
Preventive Services Task Force have both endorsed
routine HCV screening for the entire baby boomer
generation.14 Such routine screening is expected to
increase the number of people diagnosed with HCV and
provide additional opportunities for treatment intervention
and cure prior to the onset of symptomatic liver disease.
The goal at LabCorp is to support both public health
initiatives and effective drug development efforts by
offering a broad portfolio of state-of-the-art HCV tests to
ensure that existing and newly-diagnosed patients receive
the most appropriate and effective treatment regimens
LabCorp is also developing next-generation sequencing (NGS) assays to
further support the company’s HCV testing services. Currently LabCorp
offers NGS assays that provide in-depth sequencing coverage of
NS3/4A, NS5A and NS5B regions. Ultimately, the goal is to offer a single,
comprehensive NGS assay spanning the entire HCV genome. NGS assays
based on characterizations of their HCV strain and critical
host immune factors. Equipped with these tests and
new, potent direct acting antiviral therapies, healthcare
providers and agencies will be better equipped to win the
20-year battle against HCV.
are aptly suited to provide rapid, accurate and sensitive characterizations
of viral diversity within patient virus populations with the potential to
provide more in-depth and cost-effective characterizations of minority
drug-resistant mutations relative to conventional molecular cloning
procedures.
labcorp.com/clinicaltrials
5
REFERENCES
1. The Silent Epidemic: Hepatitis C. The C. Everett Koop Institute,
http://bit.ly/15849Rp (accessed August 7, 2013).
2. Mohd Hanafiah K, et al. Global Epidemiology of Hepatitis C
Virus Infection: New Estimates of Age-Speciﬁc Antibody to HCV
Seroprevalence. Hepatology (2013), 57(4):1333-42.
AUTHORS
3. Firfer H. FDA Approves New Treatment for Hepatitis C. CNN
(1999), http://bit.ly/1cuzIgY (accessed August 7, 2013).
4. Recommendations for the Identification of Chronic Hepatitis C
Virus Infection Among Persons Born During 1945–1965. World
Health Organization. MMWR: Recommendations and Reports
(August 2012), 61(4).
5. Ghany, et al. Diagnosis, Management, and Treatment of
Hepatitis C: An Update. Hepatology (2009), 49(4).
6. Hepatitis C Fact sheet N°164. World Health Organization,
http://bit.ly/novDDu (accessed August 7, 2013).
7. Hepatitis C FAQs for Health Professionals. United States
Centers for Disease Control and Prevention, http://1.usa.gov/
WTJHEd (accessed August 7, 2013).
8. Zalesak M, et al. Current and Future Disease Progression of
the Chronic HCV Population in the United States. PLoS ONE
(2013), 8(5).
9. The Hepatitis C Virus. World Health Organization, http://bit.
ly/16yzl0R (accessed August 7, 2013).
10. Ahlén G, et al. Containing “The Great Houdini” of Viruses:
Combining Direct Acting Antivirals with the Host Immune
Response for the Treatment of Chronic Hepatitis C. Drug Resist
Updat (2013), http://dx.doi.org/10.1016/j.drup.2013.06.001.
Christos J. Petropoulos, Ph.D.
Vice President and Chief Scientific Officer,
Monogram Biosciences, a LabCorp Company
[email protected]
Christos J. Petropoulos, PhD is VP of R&D and Chief Scientific
Officer of Monogram Biosciences, a LabCorp specialty
laboratory. He joined Monogram in 1996 from Genentech
where he headed the Molecular Virology Laboratory.
Dr. Petropoulos received his PhD in molecular and cell biology
from Brown University and trained as a post-doctoral fellow at
the NCI Frederick Cancer Research and Development Center.
Dr. Petropoulos has co-authored over 145 scientific journal
publications, is co-inventor on 12 issued US patents, and has
been awarded 12 small business innovative research grants
from the National Institutes of Health.
11. Ge D, et al. Genetic Variation in IL28B Predicts Hepatitis
C Treatment-Induced Viral Clearance. Nature (2009),
461:399-401.
12. Reeves J, et al. The Phenotypic Detection of HCV Polymerase
Inhibitor Resistant Subpopulations is Dependent on Relative
Resistance and Replication Capacity: IC95 and Slope Values
Can Improve Detection. Presented at the 7th International
Workshop on Hepatitis C Resistance & New Compounds, June
28-29, 2012 in Cambridge, MA.
13. Manos MM, et al. Distribution of Hepatitis C Virus Genotypes
in a Diverse US Integrated Health Care Population. Journal of
Medical Virology (2012), 84(11):1744-50.
14. Pollack A. Hepatitis C Test for Baby Boomers Urged by Health
Panel. The New York Times (2013), http://nyti.ms/133Bsdf
(accessed August 7, 2013).
PhenoSense® and GenoSure® are trademarks of Monogram Bioscienes®
6
labcorp.com/clinicaltrials
Patrice Hugo, Ph.D.
Associate Vice President and Chief Scientist,
LabCorp Clinical Trials
[email protected]
Dr. Patrice Hugo, Chief Scientist at LabCorp Clinical Trials, has
20 years of biomarker experience. He obtained his PhD at
McGill University and completed a post-doctoral fellowship at
the Walter Elisa Hall Institute in Australia and at the Howard
Hughes Medical Institute in Denver, Colorado. He was Principal
Investigator at the Montreal Clinical Research Institute and
worked in biotech operations as EVP R&D and Chief Scientific
Officer. Dr. Hugo joined the central lab industry as CSO of
Clearstone Central Lab before its acquisition by LabCorp. He is
author or co-author of over 80 scientific manuscripts.
©2014 Laboratory Corporation of America Holdings. All rights reserved.
12866-0314

Download Report