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Monogram Biosciences
Data reflected in this report are based solely on the collection of samples submitted to LabCorp for testing.
Refer to the limitations section of this report for additional guidance in interpreting the data.
Monogram Biosciences is a market leader in HIV-1 drug susceptibility testing. The assays performed at
Monogram provide genotypic and phenotypic viral resistance profiles that inform physician treatment
decisions. To date, the FDA has approved 28 antiretroviral drug/drug combinations for the treatment of
HIV/AIDS, including 7 nucleoside reverse transcriptase inhibitors (NRTI), 4 non-nucleoside reverse
transcriptase inhibitors (NNRTI), 9 protease inhibitors (PI), 5 antiretroviral drug combinations, 2 entry
inhibitors and 1 integrase inhibitor (Table 1).
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Informing optimal treatment selections for individual HIV-infected patients has positioned Monogram as a
pioneer in personalized medicine. Monogram maintains a comprehensive database of de-identified patient
HIV drug resistance test results which was used for this analysis. The drug susceptibility and co-receptor
tropism trends reported here were established by querying the Monogram database and are subject to
sampling biases inherent to Monogram’s clinical reference laboratory services, which may include, but are
not limited to, access to testing, assay clinical utility/acceptance, and reimbursement policies/practices.
Resistance to HIV-1 Protease and Reverse Transcriptase Inhibitors
Antiretroviral drug susceptibility of clinical viral samples was measured using the PhenoSenseTM HIV drug
resistance assay. The concentration of drug required to inhibit viral replication by 50% (referred to as
IC50) was determined for each patient virus, and the IC50 fold change (FC) values were calculated by
dividing the IC50 of the patient virus by the IC50 of a well-characterized, susceptible reference virus. For
the majority of antiretroviral drugs, susceptibility was assessed by comparing the FC value of the patient
virus to clinical cutoffs (Table 2),
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which are defined as the FC values that are the best discriminators of reduced clinical response based on
drug-specific clinical outcome data. A FC value below the clinical cut-off indicates the patient virus is
susceptible to inhibition by the drug, and a FC value above the clinical cut-off indicates the patient has
reduced susceptibility to the drug. Clinical cutoffs have not been established for zidovudine, nevirapine,
delaviridine, and efavirenz, therefore biological FC cutoffs were used to assess drug susceptibility Biological
cutoffs are defined as the FC value that delineate the 99th percentile of wild-type patient viruses (i.e. 99%
of WT viruses have FC values below the biological cutoff). In this report, all analyses of “drug resistance”
or “reductions in drug susceptibility” are based on the FC cutoff values listed in Table 2.
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The results from drug susceptibility testing performed at Monogram Biosciences from 2003-2009
demonstrate that the fraction of patient samples with reduced susceptibility to protease inhibitors (PI) has
been trending downward since 2007, while the fraction of samples with reduced susceptibility to nucleoside
reverse transcriptase inhibitors (NRTI) and non-nucleoside reverse transcriptase inhibitors (NNRTI) has
remained relatively stable during the same time period (Figure 1).
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This downward trend in PI resistance coincides temporally with the introduction of several additional
protease inhibitors. These PIs are potent, tolerable and select for resistance mutations that confer less
cross-resistance to other members of this drug class.
Trends in resistance to one, two and three drug classes were also evaluated (Figure 2).
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The fraction of viruses with reduced susceptibility to a single drug class is increasing. This upward trend
reflects decreases in the fraction of viruses with reduced susceptibility to PI. The prevalence of viruses with
2-drug class resistance is stable. In contrast, the prevalence of viruses with triple-drug class resistance is
decreasing over time. This downward trend also appears to be driven by a decrease in the fraction of
viruses with reduced susceptibility to PI.
Reductions in anti-HIV drug susceptibility were assessed by geographic regions within the United States in
an attempt to identify regional-specific trends (
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Figure 3A,
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Figure 3B,
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Figure 3C,
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Figure 4A,
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Figure 4B,
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Figure 4C). The West region includes states in the Pacific Time zone, the Central region includes states
from the Central and Mountain Time zones, the North-East region includes states in the Eastern Time zone
north of Virginia, and the South-East region includes states in the Eastern Time zone south of Maryland. In
general, all regional trends in HIV-1 drug susceptibility were consistent with the overall national trends, i.e.
increased single drug-class resistance and decreased triple-drug class resistance due to a reduced
prevalence of PI resistant viruses.
Trends in resistance to individual NRTI, NNRTI, and PI were evaluated by analyzing the magnitude of
reductions in drug susceptibility for patient viruses submitted to Monogram for testing from 2003-2009 (
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Figure 5A,
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Figure 5B,
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Figure 5C). FC values were calculated by dividing the susceptibility (IC50) of the patient-derived virus by
the susceptibility (IC50) of a well-characterized, susceptible reference virus. This analysis revealed a
general trend toward stable or smaller reductions in susceptibility (i.e. similar or lower levels of resistance)
with a few notable exceptions that are trending toward larger reductions in susceptibility (i.e. higher levels
of resistance). Although speculative, these trends may be driven by the availability of additional
antiretroviral agents and/or and evolving treatment practices (Table 1).
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Resistance to HIV-1 Entry Inhibitors
Susceptibility to the HIV entry inhibitor, enfuvirtide was also examined (Figure 6).
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A trend toward increased susceptibility was evident among patient samples tested from 2005-2008. The
apparent decrease in susceptibility observed in 2009 is not statistically significant when compared to the
2008 value. Patients treated with enfuvirtide are typically in the advanced stages of HIV disease and
harbor viruses that are resistant to multiple (if not all) conventional drug classes. More recently, the
treatment of such patients may have benefited by the introduction of additional treatment options that
include several antiviral drugs that target novel steps in HIV-1 replication (Table 1).
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Prevalence of Drug Resistance Mutations
The prevalence of mutations conferring reduced susceptibility to specific NRTI, NNRTI, and PI was
calculated for each drug class by year from 2003-2009. Mutation prevalence refers to the frequency of
each mutation among all viruses containing mutations associated with reduced susceptibility to any drug
class (i.e. NRTI, NNRTI, and PI). Frequencies were calculated in this manner to accurately discriminate
modest or subtle fluctuations (note that the percentages and ranges of measurements portrayed on the y
axis are specific for individual drugs). The frequencies of NRTI-associated thymidine-analog mutations
(TAM) are decreasing year to year, (Figure 7A).
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These include mutations at amino acid positions 41, 67, 70, 210, 215, and 219 of reverse transcriptase. In
addition, the NRTI-associated mutations at amino acid positions 69 and 151, which are relatively rare, but
confer resistance to the entire NRTI drug class, are also declining. The frequency of mutations at RT
position 74, which are associated with resistance to several NRTI, are also decreasing. In contrast,
mutations at RT positions 65 and 184 are increasing. NNRTI mutations at positions 100, 103, and 225 are
increasing, whereas mutations at positions 101, 106, 181, 188, and 190 are declining (Figure 7B).
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The frequencies of many mutations associated with PI resistance are decreasing, including mutations at
positions 30, 46, 47, 48, 84, and 90, whereas the frequencies of mutations at positions 50, 54, and 88 are
increasing (Figure 7C).
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Overall, the observed trends in mutation frequencies from 2003 to 2009 appear consistent with the
gradual evolution of treatment guidelines and clinical. Finally, it is important to note that the prevalence of
a given drug resistance associated mutation within the subset of viruses harboring a virus resistant to that
specific class can range dramatically; from approximately 1% to greater than 60%.
HIV-1 Replication Capacity
Replication capacity (RC) of patient-derived viruses was assessed using the PhenoSense assay by
comparing the RC of patient viruses to a well-characterized, drug susceptible HIV-1 reference virus. The
RC of the reference virus was established at 100% and the RC of a patient virus is expressed as a
percentage relative to the reference virus. RC reflects the inherent ability of a virus to replicate in the
absence of drug and can be used to assess impairments in virus replication associated with drug resistance
mutations or enhancements in virus replication associated with compensatory mutations. We found that
the RC of wild type (WT) patient viruses that exhibited susceptibility to NRTI, NNRTI, and PI remained
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similar to the reference virus from 2003-2009 (Figure 8A).
The RC of patient-derived viruses that exhibited reduced susceptibility to one or more drug classes
remained approximately 50% of the reference virus during the same time frame (Figure 8B).
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Notably, the replication capacities of both WT and drug-resistant viruses have remained stable over time.
HIV-1 Coreceptor Tropism
The TrofileTM assay was used to determine the co-receptor tropism of clinical samples originating in North
America. The TrofileTM assay was specifically developed to determine the co-receptor tropism of patient
viruses and thereby select patients that are likely to respond to treatment regimens that include specific
co-receptor antagonists (e.g. maraviroc). TrofileTM is a cell-based assay that incorporates the entire coding
region of the HIV-1 envelope protein to ensure that all envelope determinants of tropism are evaluated. By
convention, viruses that utilize the CCR5 co-receptor are referred to as CCR5-tropic (R5) viruses, viruses
that utilize the CXCR4 co-receptor are referred to as CXCR4-tropic (X4) viruses, and viruses that can utilize
CCR5 and CXCR4 are referred to as dual-tropic or mixed (DM). The initial version of TrofileTM was able to
detect minor X4 variants at ≥ 5% of the virus population, and the current enhanced version, which was
introduced in 2008, has the ability to detect X4 variants at ≤ 1% of the virus population. We found that the
proportion of R5, X4, and DM viruses has remained constant from 2007-2009 (Figure 9).
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Interestingly, the prevalence of DM and X4 viruses was higher among patient samples with resistance to
one or more drug classes (NRTI, NNRTI, and PI) (Figure 10).
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This trend may be associated with the more advanced disease stage of treatment experienced patients.
HIV-1 subtype classification was determined for clinical samples originating in North America, Europe, and
South America by nucleotide sequence determination of the gp41 transmembrane subunit of the patient
virus envelope gene (Figure 11).
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The gp41 region is more conserved than other regions in envelope, making this region more suitable for
reliable, high-throughput population-based sequencing. Subtype B comprised 97% of samples from North
America, 85% of samples from Europe, and 81% of samples from South America. In general, subtype
composition was more homogeneous in North America compared to Europe and South America, which may
reflect founder affects and immigration patterns. The prevalence of HIV-1 co-receptor tropism was
evaluated within different subtypes (clades) (Figure 12
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and Table 3).
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We report that 59% of subtype B viruses were R5-tropic, and a significantly higher prevalence of R5
viruses (compared to subtype B) were identified in subtypes A, A1, C, and G.
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