Simmering concerns about respiratory disease pandemics flared up again in mid-February after the death of a patient in Britain due to infection by a new coronavirus. The virus is part of a family which also includes the one that caused the deadly SARS (severe acute respiratory syndrome) crisis.
To recall, in the space of just seven months from November 2002, SARS spread from Hong Kong to over 37 countries, infecting over 8,000 people and killing 775. Its mortality rate was close to that of the 1918 Spanish flu outbreak – billed the ‘Mother of all Pandemics’, and 100 times more than typical influenza epidemics. SARS has since faded away, but the virus is probably lying dormant; it can also infect cats and dogs.
SARS, bird flu and swine flu
SARS outgunned the H5N1 influenza strain which also emerged out of Asia in 1997; this was largely due to the inability of the latter, best known as ‘bird flu’, to spread between people.
In 2009, another influenza strain, Type A/H1N1, involving a cocktail of genes from pigs, birds and humans, was identified in Mexico. By June, the World Health Organization (WHO) had declared the disease (dubbed ‘swine flu’) as a Level 6 pandemic , but this was due to the speed of its spread rather than mortality, which was less than the common flu.
The new coronavirus
The numbers infected by the new coronavirus are small, just 12, so far. However, the virus has some troubling characteristics. Unlike swine flu, mortality is high, and typically accompanied by pneumonia and renal failure. Of the 12 infected so far, six have died, according to the WHO. Of equal concern is the possibility of human-to-human transmission, as opposed to bird flu.
This time, the new virus has its origins in the Middle East, with Saudi Arabia and Jordan accounting for seven infections and five deaths. In November 2012, the WHO reported cases from within one Saudi Arabian family. However, it was impossible to determine if the patients were infected separately but simultaneously (during travel), or whether the disease had spread between them. Europe hosts the remaining cases – one in Germany and four in Britain, including the Birmingham fatality. While the German patient had been in Qatar, in Britain, rather than the victim, it was his father who had travelled to the Middle East. Since then, the father is reported to have infected yet another family member. Prof. John Watson, head of the Respiratory Disease Unit at the British Health Protection Agency (HPA), noted that this suggested “that person-to-person transmission has occurred.”
Nevertheless, British health officials have been quick to ward off panic. The Birmingham victim is reported to have had a weakened immune system placing him in a vulnerable risk group. The HPA’s Deputy Chief Executive Dr. Paul Cosford has underlined that the disease appears “very difficult to catch.” Prof. Wendy Barclay of Imperial College London adds: “We’re an incremental step closer to worrying, but it isn’t a worry where we need to say there is a pandemic coming.”
Getting it right
These are reassuring words for the public, but hardly so for clinical laboratories. If any of the above assumptions are (or turn out to be) wrong, the challenge for labs will be herculean – as demonstrated during the SARS crisis. Indeed, Prof. Barclay’s statements were reported four days before the new virus took its first casualty in Britain.
Though coronaviruses are fragile (they are easily destroyed by detergents and survive outside a host organism for only a day or so), the severity of illnesses like SARS compel authorities to err on the side of caution – including enforcing quarantine (with its disturbing legal implications). The nature of such a response, in turn, places inordinately heavy demands on labs to get their diagnoses right, and be ready to ramp up scale exponentially. Complicating matters further is the fact that coronaviruses are a large family. Other than SARS, they also include the virus which causes the common cold.
Though several diagnostic tests have emerged since the SARS crisis, each has its limitations. Enzyme-linked immunosorbent assays (ELISA) detect antibodies to SARS reliably, but only 21 days after the onset of symptoms. Immunofluorescence assays (IFA) take half the time but require an immunofluorescence microscope and highly skilled staff. Polymerase chain reaction (PCR) tests are extremely specific, but less sensitive: though positive results strongly indicate SARS infection, negative results do not necessarily mean its absence.
Guidelines for respiratory disease epidemics
The WHO’s laboratory guidelines for SARS hint at the magnitude of the challenge of any new respiratory disease epidemic. Above all, its recommendations on interpreting results are cumbersome. Positive PCR requires at least two different clinical specimens from a patient, or the same specimen collected on two or more days, or two different assays or repeat PCR using the original clinical sample on each occasion of testing.
For ELISA and IFA testing, the WHO specifies a negative antibody test on acute serum, followed by a positive antibody test on convalescent serum, or an over four-fold rise in antibody titre between the acute and convalescent phase
sera, which must be tested in parallel.
So far, evidence of the origin of the new Middle Eastern coronavirus is sketchy. Genetic sequencing at a Dutch laboratory has established that the virus is not the one which causes SARS. Since then, phylogenetic analysis has shown its closest relatives are bat coronaviruses from Hong Kong.
Labs: frontline defence and court of last resort
A Health Canada study titled ‘Learning from SARS’ is an excellent evaluation of the role of laboratories – above all, that of lab personnel, during the crisis. One conclusion was that though the country’s Winnipeg-based National Microbiology Laboratory (NML) was “not designed for an epidemic response”, its personnel (and those from labs across the country) managed to quickly and effectively move into crisis management mode.
The study highlighted the unique role of laboratories as both a ‘first-line’ defence against a new threat as well as a ‘court of last resort’ to improve testing – in terms of diagnosis, surveillance, and response to epidemics.
One priority, according to the Health Canada study, is to standardize testing protocols and share data, to “see the whole picture” of an evolving epidemic. This, it argued, required laboratory information systems (LIS) that are “agile, modular, and rapidly modifiable for special purposes“, a lesson which has relevance for LIS designers even today. On its part, the WHO has mentored an international network of laboratories to identify best practices from the SARS
experience. This will clearly have a bearing on preparations for any new epidemic.
The impact of air travel
The challenge of respiratory system viruses is emphasized by the huge numbers of air travellers. Though little research has been done on the role of airplanes in respiratory epidemics, circumstantial evidence is strong. When SARS struck, 16 of 120 people on a single flight from Hong Kong to Beijing developed the disease, from just one index case. Conversely, the fall in air travel after the September 2011 US terror attacks sharply reduced flu incidence during the year.
Today, of some 8 million air passengers aloft every day, over 1 million cross international borders, just like the victims of the new virus in Britain and Germany. This is an area clearly in need of official attention. Indeed, in March 2003, the WHO recommended screening airline passengers for SARS but its impact was minimal, and questionable. Given the massive number of air travellers, it is clear that any new respiratory epidemic will first grow by leaps and bounds before any meaningful steps can be devised to control it.
The promise of biosensors
Some experts believe that airports should be provided with the means (and the authority) to screen passengers in an impending epidemic, for alternative causes. During the SARS crisis, such eliminative tests – even in a sophisticated setting like the US – were “ordered at the discretion of local clinicians”, diagnosed on “the basis of local interpretations” and many “were never reported to CDC.”
Today, at least one handheld, biosensor-based kit for diagnosing influenza A and B and respiratory syncytial viruses (RSV) – without having to send samples to the lab – is close to market. Deploying such devices at airports ought to be the next step, given the potential threat from the new Middle Eastern coronavirus as well as others that may arise in the future.
This would free laboratories to concentrate on their main task – to identify and confirm genuine, high-risk cases and direct their expertise to what Health Canada billed as their role as a ‘court of last resort’: to quickly master new diagnostic techniques and ensure a quicker response to containing epidemics.
Interindividual variability in the response to clopidogrel has been shown to be related to the clinical ischemic outcomes. Although testing of platelet function or genetic profile is recommended to evaluate the response to clopidogrel, standardized testing and definitive antiplatelet therapy after testing need to be established.
by Yusuke Yamaguchi and Professor Mitsuru Murata
Clinical background
Platelet activation and aggregation play a pivotal role in arterial thrombosis formation; therefore, antiplatelet therapy to inhibit platelet function is considered effective for preventing and treating atherothrombosis. The combination of aspirin and clopidogrel has been shown to be more effective than aspirin alone for improving clinical ischemic outcomes in patients with coronary artery disease (CAD). This dual antiplatelet therapy contributes substantially to prevent the occurrence of cardiovascular events in patients with acute coronary syndrome (ACS) or percutaneous coronary intervention (PCI). Current guidelines recommend aspirin and clopidogrel for these patients; however, some patients still develop cardiovascular events despite dual therapy. It has been shown in the last decade that the responsiveness to clopidogrel is highly variable in individuals and that a suboptimal response to clopidogrel is a risk factor for cardiovascular events. The interindividual variability in the effect of clopidogrel is due to multiple factors [Table 1].
Effects of CYP2C19 on clopidogrel
Clopidogrel, a second generation thienopyridine, is an inactive prodrug that requires a 2-step metabolic conversion to an active metabolite. This active metabolite inhibits adenosine diphosphate (ADP)-induced platelet aggregation by selectively and irreversibly binding P2Y12 receptors on the platelet membrane. Several isoforms of cytochrome P450 (CYP), including CYP2C19, CYP3A4, CYP1A2, CYP2B6, and CYP2C9, have been shown to be involved in the metabolic pathway. Of these enzymes, CYP2C19 is considered to be the main determinant of clopidogrel metabolism that produces the active form.
It is known that CYP2C19 has numerous single nucleotide polymorphisms (SNPs), of which CYP2C19*2 (681G>A, located in exon 5) has been studied extensively and shown to be associated with a loss of function of the enzyme. CYP2C19*2 clearly associates with both the pharmacokinetics (i.e., area under the concentration curve and maximal plasma concentration of clopidogrel active metabolite) and the pharmacodynamics (i.e., inhibition of ADP-induced platelet aggregation) of clopidogrel. CYP2C19*2 is detected more frequently in Asians than in Caucasians, with approximately 40–50% and 30% having at least one CYP2C19*2 allele, respectively. In addition to CYP2C19*2, CYP2C19*3, *4, *5, *6, *7, and *8 have been identified as loss-of-function alleles.
Methods to evaluate the effect of clopidogrel on platelet inhibition
Different laboratory tests [Table 2] can be used to assess platelet function in patients treated with clopidogrel. ADP-induced platelet aggregation in platelet-rich plasma measured by light transmission aggregometry is used most commonly, with numerous published studies using this method to measure platelet function. The majority of these studies measured platelet function as maximal platelet aggregation rate induced by 5, 10, or 20 µmol/l ADP. The platelet aggregation rate 5–8 min after the addition of ADP has also been reported. The POPULAR study [1] on clopidogrel-treated patients following elective PCI showed that 42.9% maximal platelet aggregation rate induced by 5 µmol/l ADP or 64.5% induced by 20 µmol/l ADP correlated with the 1-year mortality rate, myocardial infarction (MI), stent thrombosis, and stroke.
The VerifyNow P2Y12 test (Accumetrics Inc, SanDiego, CA) has been developed as a point-of-care device to quickly and accurately assess platelet function in patients. This test is a whole-blood, light transmission-based optical detection assay that measures the light transmittance of ADP-induced platelet aggregation in a cartridge containing fibrinogen-coated beads and is able to specifically evaluate P2Y12 receptor inhibition. The results are reported as P2Y12 reaction units (PRU), with a lower PRU value being associated with higher P2Y12 inhibition. A meta-analysis of individual patient data in six observational studies [2] revealed that a PRU value of 230 at PCI is the best cut-off value for predicting the occurrence of cardiovascular events, including death, MI, and stent thrombosis, in patients with stable CAD or non-ST elevated ACS undergoing PCI over 1 year.
The effect of clopidogrel on platelet function can be also evaluated by detecting vasodilator-stimulated phosphoprotein (VASP). VASP is phosphorylated by cyclic adenosine monophosphate (cAMP) produced in the adenylate cyclase cascade downstream of the P2Y12 receptor. By binding to the P2Y12 receptor and suppressing the cascade, ADP leads to an increase in VASP dephosphorylation, whereas inhibition of the receptor by clopidogrel active metabolite leads to an increase in VASP phosphorylation. This test measures VASP phosphorylation in a flow cytometric assay with the result expressed as platelet reactivity index (PRI) that represents the ratio of the phosphorylated and dephosphorylated VASP. A lower PRI value reflects higher P2Y12 inhibition.
Clinical utility of laboratory testing
Numerous studies, including our meta-analysis [3], have reported that patients with a suboptimal response to antiplatelet therapy have increased cardiovascular events [Figure 1A], and data have been accumulated on testing of platelet function to establish a reliable cut-off value for clinical risk. However, it remains unclear how to monitor suboptimal responses in daily clinical practice due to the lack of a standardised method to measure and interpret the results of platelet function. Furthermore, there is no guideline for alternative treatment strategies to the “one-size-fits-all” 75 mg/day clopidogrel regime because conclusive evidence that personalised antiplatelet therapy improves patient outcomes has not been established from large-scale randomised trials. However, a meta-analysis [4] recently reported the evaluation of the clinical efficacy and safety of intensified antiplatelet therapy involving reloading clopidogrel, using glycoprotein IIb/IIIa inhibitors periprocedural PCI, increasing the maintenance dose of clopidogrel, or switching to prasugrel. Although there were several limitations, this meta-analysis showed that intensified antiplatelet therapy reduces cardiovascular death and stent thrombosis without increasing major bleeding.
Meanwhile, CYP2C19 genotype does not always seem to predict cardiovascular events, although it is a major predictor for suboptimal response to clopidogrel. To date, many large-scale clinical trials, including the recent Genotype Information and Functional Testing (GIFT) trial [5], which investigate an association of CYP2C19 genotype with cardiovascular events, have been performed. However, the results of these trials were inconsistent. Indeed, we showed heterogeneity in the odds ratio of the cardiovascular events between the carriers and non-carriers of CYP2C19*2 allele in our meta-analysis [Figure 1B]. Considering that CYP2C19*2 contributes to only about 5% of the variability in response to clopidogrel [6], many other genetic factors may contribute to the variability apart from CYP2C19. Therefore, genetic testing including additional factors such as SNPs in other CYPs or ABCB1 (encoding p-glycoprotein) would be expected to improve identification of patients with a suboptimal response.
Current status and future prospects
In 2009, the U.S. Food and Drug Administration (FDA) released a black box warning that significant attention needs to be paid to clopidogrel pharmacogenomics. Similarly, the American and European guidelines published in 2011 gave a Class IIb recommendation for testing of platelet function or genetic profile in patients treated with clopidogrel and for consideration of the use of an alternate P2Y12 inhibitor in patients with inadequate platelet inhibition.
The primary goal of testing of platelet function and genetic profile is to identify patients with a suboptimal response to antiplatelet therapy and provide them with a tailor therapy to improve the clinical ischemic outcomes without an
associated bleeding risk. Although these laboratory tests provide sufficient evidence to predict outcomes, personalised antiplatelet therapy on the basis of these tests has not been established in the guidelines. Currently, several clinical trials are ongoing that evaluate the effect of personalised antiplatelet therapy on the basis of laboratory tests. These trials will hopefully provide important data to establish guidelines, to allow clinicians to properly select laboratory tests, and to plan personalised antiplatelet therapy in patients with a suboptimal response.
References
1. Breet NJ, van Werkum JW, Bouman HJ, Kelder JC, Ruven HJ, Bal ET, et al. JAMA 2010; 303: 754–762.
2. Brar SS, ten Berg J, Marcucci R, Price MJ, Valgimigli M, Kim HS, et al. J Am Coll Cardiol 2011; 58: 1945–1954.
3. Yamaguchi Y, Abe T, Sato Y, Matsubara Y, Moriki T, Murata M. Platelets. Epub 2012 Jul 3, doi: 10.3109/09537104.2012.700969
4. Aradi D, Komócsi A, Price MJ, Cuisset T, Ari H, Hazarbasanov D, et al. Int J Cardiol. Epub 2012 Jun 15, doi: 10.1016/j.ijcard.2012.05.100
5. Price MJ, Murray SS, Angiolillo DJ, Lillie E, Smith EN, Tisch RL, et al. J Am Coll Cardiol 2012; 59: 1928–1937.
6. Hochholzer W, Trenk D, Fromm MF, Valina CM, Stratz C, Bestehorn HP, et al. J Am Coll Cardiol 2010; 55: 2427–2434.
The authors
Yusuke Yamaguchi and Mitsuru Murata MD, PhD
Dept of Laboratory Medicine, Keio University School of Medicine,
35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan
E-mail: yusukeyamaguchi@z8.keio.jp
Pertussis (whooping cough) has been a significant cause of morbidity and mortality in young children since the first epidemic was described in 1578. Currently in the West even when infants suffering from the disease are hospitalized and appropriately treated, around 1% still die, and in less developed countries the mortality rate in infants is as high as 4%. However, following the isolation of the causative organism Bordetella pertussis over a century ago, years of research and development resulted in the introduction of an effective vaccine in the 1940s.
The whole cell vaccine used heat-killed bacteria combined with diphtheria and tetanus toxoids to give the classical DPT vaccine, usually given to infants three times during their first year of life with further booster doses twice during childhood. The advent of this vaccine did not prevent the three to five year pertussis epidemic cycle, but it elicited a strong immune response and the total number of cases plummeted in immunized populations. There were some common side-effects, including swelling, mild fever and pain, but these were trivial compared with the high risk of children contracting pertussis if they were not immunized. Sadly, though, very dubious research linked cases of SIDS and encephalopathy with use of whole cell pertussis vaccine, and the popular press eagerly disseminated this dangerously misleading information. Parents began to exercise their so-called ‘freedom of choice’ based on a dearth of unbiased information and stopped having their children immunized, so in the 1990s a new acellular vaccine (DPaT) with fewer side effects gradually replaced the classical DPT.
Now cases of pertussis have more than tripled in the last five years in much of the globe, and the resulting whooping cough epidemic is the worst for 50 years. While it is possible that a more virulent strain of bacterium has evolved, the most likely explanation is that the ‘new’ vaccine is not as effective as its predecessor. Indeed a recent robust study from Australia compared incidence of pertussis in 40,694 children who were immunized in 1998 with either DPT or DPaT (both vaccines were still in use at that time). Significantly higher rates of pertussis were found in the children who had received the latter vaccine.
The suggested solution to the pertussis epidemic is to extend immunisation programmes to cover pregnant women as well as all those who come in contact with young infants. Wouldn’t reintroducing the old vaccine be simpler?
The ability to use genetic information to inform clinical decision making has emerged as a new tool in clinical practice, with noteworthy examples across many area of medicine. Cardiology and anticoagulation in particular have led the way in the translation of genetic findings into actionable clinical recommendations, spurred by the addition of genetically guided dosing in the drug label for warfarin by the FDA. This review covers the pharmacogenomics related to warfarin therapy.
by Dr Minoli Perera
Pharmacogenomics is primarily aimed at identifying genetic variation that influences inter-individual differences in drug response. The guiding principle is “the right drug, at the right dose for the right person”. Its application promises to enable targeted drug administration, improve therapeutic outcomes, and inform drug development. Pharmacogenomic insights have also improved our understanding of the underlying pathways and mechanisms behind adverse drug reactions. Such adverse reactions account for approximately 100 000 deaths per year in the US, and markedly increase healthcare costs. Advances made over the last 30 years in molecular biology, molecular medicine, and genomics have had a major impact on the development of pharmacogenomics.
Currently a variety of approaches are used to associate genetic variants associated with drug response. Commonly used strategies include candidate gene approach and genome-wide association studies (GWAS). Candidate gene studies investigate single nucleotide polymorphisms (SNPs) that are correlated to drug response. These studies are usually restricted to genes or SNPs that have been shown to be involved in the pathway of drug action or drug clearance. Genome-wide association studies investigate up to 5 million SNPs spaced throughout the genome which are genotyped to identify genetic variants with the drug phenotype in an unbiased fashion. Each of these methods has advantages and disadvantages. Genome-wide studies comprehensively cover the entire genome, but their power to detect moderate associations is greatly limited by the multiple testing burden, which is a requirement for correction for false-positive associations. The candidate gene approach narrows the focus to a few important SNPs and therefore has higher power, but may miss the real causative SNP, and require a priori knowledge for the selection of SNPs/genes to study.
Both these methods have yielded important clinical findings that can immediately be used to reduce the incidence of adverse effects (many of which have been added to drug labels). Notable examples such as the SLCO1B1*5 polymorphism (associated with myopathy with statin use) and CYP2C19*2 (associated with clopidogrel non-response) have shown clinically meaningful outcomes related to genetic variants. However, the translation of these findings has been slower in coming and has many clinicians wondering about the utility of this new technology.
The “test case” for pharmacogenetics was thought to be pharmacogenetically guided warfarin dosing. In this review we will cover the genetic polymorphism effecting warfarin dose requirements and the currently available diagnostic tests, as a case study for the implementation of pharmacogenetics in the clinic.
Genes that affect warfarin dose
Numerous studies, predominately conducted in Caucasian and Asian populations, demonstrate that the CYP2C9 and VKORC1 genotypes contribute significantly to warfarin dose variability [1–3]. The role of these gene products can be seen in Figure 1. The CYP2C9 enzyme metabolises the more active S-enantiomer of warfarin to inactive 7-hydroxywarfarin. Warfarin inhibits the enzyme VKOR (encoded by the gene VKORC1) to prevent conversion of Vitamin K epoxide to its reduced form necessary for activation of the clotting factors II, VII, IX and X. Thus, SNPs in the CYP2C9 gene affect warfarin pharmacokinetics, whereas variation in the VKORC1 gene impacts warfarin pharmacodynamics.
The most extensively studied CYP2C9 variants are the CYP2C9*2 and CYP2C9*3 alleles, which lead to significant reductions in CYP2C9 activity. Compared with the people without this genotype, carriers of the *2 or *3 genotypes have S-warfarin clearance reduced to between 40 to 90% or normal levels. As a result, significantly lower doses are usually needed in individuals with a CYP2C9*2 or CYP2C9*3 allele. The CYP2C9 genotype is also implicated in the risk of bleeding during warfarin therapy, especially during the warfarin initiation period [4].
The VKORC1 genotype was originally recognised for causing warfarin resistance through mutations in the gene-coding region. More recently, common VKORC1 SNPs occurring in gene-regulatory regions and underlying usual warfarin dose variability were discovered [3]. Two such SNPs, one in the promoter region (-1639G>A) and one in intron 1 (1173C>T), show the strongest association and possible functional effects [5]. Thus, the majority of warfarin pharmacogenetic studies have focused on one of these two SNPs, which are in strong linkage disequilibrium across populations (meaning they are inherited together and strongly associated with each other). This means that only one of these SNPs needs to be taken into account for pharmacogenetic dosing of warfarin. Most investigators chose the VKORC1 -1639G>A as the predictive SNP in warfarin pharmacogenetic studies. This SNP explains approximately 20–28% of the overall variability in dose requirements in Caucasians, but only 5–7% of the variability in African–Americans, mainly due to the difference in allele frequency between populations [6, 7]. Unlike CYP2C9, the VKORC1 genotype does not appear to affect the risk of bleeding with warfarin treatment[4].
These findings have been confirmed in several genome-wide association studies (GWAS) in Caucasians and Asian individuals, showing that VKORC1 -1639G>A, CYP2C9*2 and CYP2C9*3 polymorphisms are the primary genetic determinants of warfarin dose requirements in these populations. The combination of VKORC1 -1639G>A, CYP2C9 (*2 and *3) and clinical factors (e.g., age, sex, weight and amiodarone use) explains approximately 55% of the total variance in warfarin maintenance dose in Caucasians, but only about 25% among African–Americans. With the exception of the CYP4F2 genotype, found in a GWAS study of Swedish patients [8], no other genetic variant has met genome-wide significance for association with warfarin dose requirements. Both genetic and non-genetic variables have been included in dosing algorithms that can be used to predict dose, such as WarfarinDosing.org.
Warfarin genetic testing and guidelines
Insurance companies consider genetic testing for genetic variants in CYP2C19 related to clopidogrel response and the HLA-B* 1502 allele for prediction of adverse effects related to carbamazepine as “medically necessary” and may therefore cover the cost of these tests. The same does not hold for warfarin testing, which is considered investigational and will only be covered in the context of a clinical trial.
In 2007, the US Food and Drug Administration modified the package insert for warfarin to include information on the relationship of safe and effective dosage to SNPs in CYP2C9 and VKORC1, including a table of recommended doses for each genotype combination [Table 1]. These recommendations give the range of doses that should be considered when dosing a patient that is a carrier of any of the tested SNPs. However, there is increasing evidence that additional alleles outside of CYP2C9*2 and *3 and VKORC1 -1639G/A may play a role in warfarin dose response. These SNPs are not included in the FDA dose recommendations and not all tests cover all these additional variants.
Currently, four warfarin pharmacogenetic tests are available as in vitro diagnostic devices [shown in Table 2]. All of these tests genotype for three loci: CYP2C9*2, CYP2C9*3 and one VKORC1 -1639G/A or 1173C/T (both of which give equivalent information because of the afore mentioned LD in all populations), with some including other known genetic variants that are associated with warfarin dose. All of the tests can be completed in 8 hours, including DNA extraction, with the fastest ones providing genotype results in less than 2 hours.
The Clinical Pharmacogenetics Implementation Consortium (CPIC) recently published guidelines on how to interpret and apply genetic test results to adjust warfarin doses [9]. These guidelines do not address when to order a genetic test, but rather how to dose warfarin when genetic test results are available. The guidelines strongly support the use of genetic information to guide warfarin dosing when genotype is known and recommend using either the International Warfarin Pharmacogenetics Consortium (IWPC) or Gage algorithm to do so.
Although the availability of FDA-cleared devices for warfarin pharmacogenetic testing makes genotype-guided warfarin initiation possible, several barriers to clinical adoption remain. First, many medical centres do not have warfarin pharmacogenetic testing available. In a recent survey, only 20% of hospitals in North America have testing available on site, suggesting the majority of the hospitals rely on outside commercial clinical laboratories. This outsourcing may make genotype-guided warfarin initiation impractical because of 3–7 days of turnaround time. Second, no professional organisation endorses warfarin pharmacogenetic testing in its guidelines because of the lack of the clinical utility data. Inclusion of a testing recommendation in professional guidelines has been identified as a factor influencing reimbursement of new technology. As such, the Centers for Medicare and Medicaid Services (CMS) and many commercial insurance plans generally do not reimburse the cost of testing ($300–500). Because of these barriers, warfarin pharmacogenetic testing is performed mainly for research purposes and for patients willing to pay the cost.
Future perspective and conclusions
There are substantial and convincing data supporting the clinical and analytic validity of warfarin pharmacogenetics. The CYP2C9 and VKORC1 genes are the primary determinants of warfarin dose requirements. There are several FDA-cleared tests available for CYP2C9 and VKORC1 genotyping. However, genotype-guided warfarin dosing has not yet become a reality in most medical centres despite the wealth of data supporting genetic influences of warfarin dose requirements. Many clinicians and third party payers are awaiting evidence of clinical utility and cost-effectiveness before adopting genetic testing for anticoagulation management in the clinic setting. Results from ongoing clinical trials (such as the NIH-sponsored COAG trial) are expected to address these issues and will likely determine the course of genotype-guided anticoagulant therapy. Whether pharmacogenetics will have a role in the treatment with newer anticoagulant agents has yet to be determined. However, the pharmacogenetics with these anticoagulants could be of great importance given the unavailability of routine monitoring parameters with these agents.
References
1. Wadelius M, et al. Blood 2009; 113: 784–792.
2. Klein TE, et al. N Engl J Med 2009; 360: 753–764.
3. Rieder MJ, et al. N Engl J Med 2005; 352: 2285–2293.
4. Limdi NA, et al. Clinical pharmacology and therapeutics 2008; 83: 312–321.
5. Wang D, et al. Blood 2008; 112: 1013–1021.
6. Cavallari LH, et al. Clin Pharmacol Ther 2010; 87: 459–464.
7. Perera MA, et al. Clin Pharmacol Ther 2011; 89: 408–415.
8. Takeuchi F, et al. PLoS Genet 2009; 5: e1000433.
9. Johnson JA, et al. Clinical pharmacology and therapeutics 2011; 90: 625–629.
10. Coumadin package insert. 2007. (Accessed October, 2007, at http://www.bms.com/cgi-bin/anybin.pl?sql=PI_SEQ=91.)
The author
Minoli A Perera, PharmD., PhD
Knapp Center for Biomedical Discovery
Room 3220B, University of Chicago, 900 E.
57th Street, Chicago, IL 60637, USA
E-mail: mperera@bsd.uchicago.edu
Primary hepatic tumours are one of the most aggressive and resistant forms of cancer. Early diagnosis of liver cancer and the development of more accurate markers for biological classification are crucial to improving the clinical management and survival of patients. This article discusses the emerging use of microRNAs for the diagnosis of liver cancer.
by Dr Luc Gailhouste and Dr Takahiro Ochiya
Liver cancer and diagnosis
Primary liver cancer is mainly represented by hepatocellular carcinoma (HCC) and accounts for almost 90% of primitive hepatic malignancies. Statistically, HCC is the third most common cause of death from cancer worldwide [1] and is generally encountered in patients exhibiting an underlying chronic liver disease such as hepatitis B virus (HBV) and/or C virus (HCV) infection, alcohol abuse, or liver steatosis. Chronic hepatitis leads to fibrosis and gradually evolves into cirrhosis. Global studies estimate that approximately 80–90% of all HCCs arise from cirrhotic livers. Despite great advances in the treatment of the disease, hepatic cancer exhibits one of the lowest remission rates (less than 10% after five years), mainly due to its late diagnosis and high resistance to the conventional agents of chemotherapy. Indeed, as such a disease tends to remain asymptomatic, approximately 50% of newly diagnosed patients already exhibit late advancement.
Common HCC diagnostic methods include liver imaging techniques such as triphasic computed tomography scanning, magnetic resonance imaging (MRI), and abdominal ultrasound [2]. A panel of serological biochemical markers, including aminotransferases ALAT and ASAT, has also been used for several decades to monitor liver pathologies in a non-invasive manner.
Until recently, imaging tests were frequently combined with the non-invasive measurement of serum alpha-fetoprotein (AFP). Normally produced by the fetal liver, AFP decreases soon after birth whereas its high level in adults can be correlated with the appearance of malignant hepatic disease. However, the American Association for the Study of Liver Diseases (AASLD), in its practice guidelines, discontinued the use of the blood tumour marker AFP for surveillance and diagnosis due to the limited sensitivity and specificity of the method. When uncertainty regarding the diagnosis persists, a percutaneous biopsy followed by histological examination of the nodule is indicated [3]. This technique remains the gold standard method for determining the degree of underlying fibrosis and shows appreciable sensitivity (more than 80%) for HCC diagnosis.
An important breakthrough in the clinical management of liver cancer would come from the accurate correlation of the alterations of cancer-related genes and the tumour phenotype. Although HCC lesions can be broadly distinguished by histological or immunological assessment, their prognosis and clinical evolution vary greatly from one individual to another. The discovery of innovative and effective biomarkers ensuring an early diagnosis of the disease correlated with the etiology, the pathogenic tendency, and the malignancy of the tumour could significantly enhance the molecular assessment of HCC and its classification in order to maximize the positive response of therapeutics.
MicroRNAs: biogenesis and mechanism of action
MicroRNAs (miRNAs) constitute a group of evolutionary conserved small non-coding RNAs of approximately 22 nucleotides that accurately regulate gene expression by complementary base pairing with the 3’-untranslated regions (3’-UTRs) of messenger RNAs (mRNAs) [4]. These post-transcriptional regulators were first evidenced in C. elegans by Ambros and co-workers who discovered that lin-4, a gene known to control the timing of nematode larval development, did not code for a protein but produced small RNAs that specifically bind to lin-14 mRNA and repress its translation.
miRNA biogenesis is a multistep process that has been reviewed extensively [Figure 1]. An essential feature of miRNAs is that a single miRNA can recognize numerous mRNAs, and, conversely, one mRNA can be recognized by several miRNAs. These pleiotropic properties enable miRNAs to exert wide control over a plethora of targets, attesting to the complexity of this mechanism of gene expression regulation. Several reports have described the key role of these post-transcriptional regulators in the control of diverse biological processes such as development, differentiation, cell proliferation, and apoptosis. The alterations of miRNA expression have also been reported in a wide range of human diseases, including cancer [5].
In HCC, the atypical expression of miRNAs frequently contributes to the deregulation of critical genes known to play an essential role in tumorigenesis and cancer progression. The current consensus is that cancer-related miRNAs function as oncogenes or tumour suppressors [6]. As for other malignancies, two situations can occur in HCC: (i) tumour suppressor miRNAs can be downregulated in liver cancer and cause the upregulation of oncogenic target genes repressed in normal hepatic tissues, increasing cell growth, invasion abilities, or drug resistance; (ii) oncogenic miRNAs, also called oncomirs, can be upregulated in HCC and can downregulate their target tumour suppressor genes, potentially leading to hepatocarcinogenesis.
miRNA as a diagnostic tool
As miRNA signatures are believed to serve as accurate molecular biomarkers for the clinical classification of HCC tumours, the availability of consistent technologies that enable the detection of miRNAs has become of interest for both fundamental and clinical purposes. The most current detection methods commonly used are microarray and real-time quantitative polymerase chain reaction (RT-qPCR).
Microarray analysis presents the advantage of offering a high speed of screening by employing various miRNA probes within a single microchip. However, the technique has lower sensitivity and specificity than RT-qPCR, which is the most widely used method.
miRNA RT-qPCR is based on the use of stem–loop primers, which can specifically bind to the mature miRNA during reverse transcription, granting a high degree of accuracy to the method [7]. Analysis of miRNAs by RT-qPCR is a cost-effective technique and, due to its efficiency, a valuable way to validate miRNA signatures. Moreover, the development of RT-qPCR protocols has improved the sensitivity of miRNA detection down to a few nanograms of total RNAs. This amount can be easily and routinely obtained by extracting total RNAs from a small fragment of a hepatic percutaneous biopsy.
A plethora of studies have already reported various miRNA profiles potentially reflecting HCC initiation and progression that could be employed as specific cancer biomarkers [8]. Comparative analysis of bibliographic data provides evidence of the persistent augmentation of miR-21 in cancer, regardless of the tumour origin. In the HCC, miR-21 is also frequently overexpressed where it acts as an oncogenic miRNA. The major overexpression of miR-21 is associated with the inhibition of the tumour suppressor PTEN and the poor differentiation of the tumour. The use of an miRNA-based classification correlated with the etiology and the aggressiveness of the tumour appears very promising, as it could significantly enhance the accuracy of the molecular diagnosis of HCC and its classification, leading to the consideration of more appropriate therapeutic strategies.
In this regard, Budhu and collaborators defined a combination of 20 miRNAs as an HCC metastasis signature and showed that this 20-miRNA-based profile was capable of predicting the survival and recurrence of HCC in patients with multinodular or single tumours, including those at an early stage of the disease [9]. Remarkably, the highlighted expression profile showed a similar accuracy regarding patient prognosis when compared to the conventional clinical parameters, suggesting the relevance of this miRNA signature. Consequently, the profiling of aberrantly expressed cancer-related miRNAs might establish the basis for the development of a rational system of classification in order to refine the diagnosis and the prediction of HCC evolution.
Tumour suppressor miRNA: the case of miR-122
The case of miR-122 is of prime interest, first, because it represents by itself more than half of the total amount of miRNAs expressed in the liver [10]. Remarkably, miR-122 is a key host factor required for HCV replication. A phase 2 clinical trial was recently initiated that reported the world’s first miRNA-based therapy targeting miR-122 in HCV-infected patients using the locked nucleic acid (LNA)-modified antisense oligonucleotide miravirsen [11]. Thus, a four-week miravirsen treatment by subcutaneous injection provided long-lasting antiviral activity and was well tolerated.
However, the experimental silencing of miR-122 resulted in increased expression of hundreds of genes normally repressed in normal hepatocytes. The miR-122 knockout mouse model displays hepatosteatosis, fibrosis, and a high incidence of HCC, suggesting the tumour suppressor role of miR-122 in the liver. In primary liver carcinoma, the existence of an inverse correlation was demonstrated between the expression of miR-122 and cyclin G1, which is highly implicated in cell cycle progression.
Regarding the potential of miR-122 as a diagnostic biomarker in liver cancer, numerous studies have already reported the significant and specific downregulation of miR-122 expression in both human and rodent HCC models. Obviously, miR-122 was shown as downregulated in more than 70% of the samples obtained from HCC patients with underlying cirrhosis as well as in 100% of the HCC-derived cell lines [12].
To illustrate this statement, we analyzed the expression levels of miR-122 in 20 patients who exhibited HCC using RT-qPCR. Following RNA extraction from biopsies with the miRNeasy Mini Kit (Qiagen), 100 ng of total RNA was reverse-transcribed using the Taqman miRNA Reverse Transcription Kit (Applied Biosystems). The expression levels of mature miR-122 were determined in each sample by RT-qPCR with Taqman Universal PCR Master Mix in a 7300 Real-Time PCR System from Applied Biosystems. The expression levels of miRNAs were normalized with respect to the endogenous levels of RNU6B. RT-qPCR data were obtained easily and rapidly by a routinely conventional method used in our laboratory. As a result, miR-122 expression was reduced more than threefold in HCC biopsies relative to the normal liver group (median 0.935 and 3.495, respectively; P<0.0001, Mann–Whitney U test) [Figure 2]. These data suggest that cancer-related miRNAs, such as miR-122, which are deregulated in HCC tissues, could be relevant with regard to the development of new diagnostic tools and the clinical management of liver cancer patients.
Conclusions and emerging approaches
The expression profile of specific miRNAs has been found to reflect the biological behaviour of HCC tumours, such as aggressiveness, invasiveness, or drug resistance. As a consequence, miRNA investigations may offer opportunities to determine miRNA signatures that would provide valuable information to stratify and refine HCC diagnosis in terms of prognosis, response to treatment, and disease relapse. Recently, tumour-derived miRNAs have been efficiently detected in the serum of patients and characterized as potential non-invasive biomarkers for HCC.
The concept that miRNAs could serve as potential plasma markers for liver diseases is, thus, gaining attention. Due to its frequent deregulation in viral hepatitis, cirrhosis, and cancer as well as its specific and massive expression in the liver, the assessment of serum miR-122 could represent one reliable strategy for the non-invasive diagnosis of chronic liver pathologies. Although the process of assessing serum miRNAs remains under improvement, cancer-related circulating miRNAs represent an exciting and promising field of investigation for the development of more accurate technologies for the early diagnosis of HCC.
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The authors
Luc Gailhouste PhD and
Takahiro Ochiya PhD
Division of Molecular and Cellular
Medicine, National Cancer Center Research Institute, Tokyo, Japan
November 2024
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The Netherlands
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info@clinlabint.com
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