Essay: Warafin genotyping in patients

WARFARIN GENOTYPING IN PATIENTS USING CONTINUOUS FLOW LEFT VENTRICULAR ASSIST DEVICES
 
INTRODUCTION
In the U.S., and increasingly around the world, heart failure (HF) is responsible for a large number of hospital admissions, high mortality rates and a high cost of care. According to the Center for Disease Control and Prevention (CDC), there was a promising decline in the number of deaths associated with HF from 2000 to 2012 in the United State but this was followed by a slight and steady increase in heart failure deaths through 2014 (1). Recent and continuous advancement in research, diagnosis and treatment of heart failure, however, has placed this increase in check. Some of the improvements in diagnosis and treatment include cardiac catheterization and nuclear medicine, with echocardiography considered the most practicable way of accessing cardiac function (2). In individuals with heart failure, the ventricles, which pump blood to the body, are inefficient in either filling or ejection, resulting in an inadequate blood flow (3). This condition may be caused by a number of factors including hypertension, diabetes mellitus, metabolic syndrome, familial cardiomyopathies or age (3).
The New York Heart Association (NYHA) and the American College of Cardiology Foundation (ACCF) have classified heart failure into varying ranges of severity based on functional capacity and disease development and progression (4). The classification ranges from class I to class IV, with class I indicating no limitation of physical activity, while class IV indicates that the patient shows severe symptoms of the disease even at rest (4). Several studies have shown that the absolute mortality rate of individuals who are diagnosed with HF remains 50% within 5 years (3). This rate increases as the condition of the patient worsens from class I to class IV, and results in a survival rate of 97% and 20% respectively (3).
The improvement in treatment methods employed for heart failure patients have resulted in an increase in the number of patients who reach Class IV HF, which is also considered refractory or end-stage HF. These class IV patients also have a 1-year mortality rate of 50% (5). To maximize the treatment efficacy of this patient population, a combination of pharmacological, surgical and other therapeutic treatments are used. Angiotensin Converting Enzyme (ACE) inhibitors or angiotensin II type I receptor blockers (ARBs) and other drugs like Beta-adrenergic receptor blockers have been shown to be effective at improving clinical outcomes of HF especially at early stages (3-5). When conditions permit however, end-stage HF patients may be placed on a ventricular assist device as they transition to cardiac transplant (BTT), or permanently as a destination therapy (DT).
Heart transplants are considered the gold standard of care but have gradually declined due to low availability of donor hearts while the use of mechanical assist devices continues to grow. Research has proven that the continuous-flow left ventricular assist device (CF-LVAD) significantly improves the quality of life and the functional capacity of these patients (6). The CF-LVAD works by shunting blood from the left ventricle through a pumping tube that continuously directs blood flow to the aorta. The latter was a pulsatile device that mimicked the pulsating rhythm of the heart but was discontinued when it was observed to have no improvement in HF patient condition (7). Despite the effectiveness of the CF-LVAD, patients reportedly experienced device induced stroke and bleeding events (7). Crow et al concluded from their study, that 63 out of every 100 patient on CF-LVADs show long-term gastrointestinal bleeding (7). A possible explanation for these bleeding and thrombotic events includes loss of von Willebrand factors (vWF) due to high shear stress from the implanted heart device device (8). It is understood that controlling coagulating factors via the intake of vitamin K antagonist like warfarin, and platelet inhibitors like aspirin, substantially limits the problem of clotting. Nevertheless, variations in the response of patients to warfarin dosage and fluctuating INR values out of the therapeutic range clouds the efficacy of this approach (8).
Warfarin is a derivative of coumarin that works by inhibiting the activity of the vitamin k epoxide reductase enzyme (VKOR). It does this by preventing the post-translational carboxylation of certain vitamin k dependent clotting factors including factors II, VII, IX, X and proteins C and S (9). The VKOR enzyme is responsible for activating and regenerating vitamin k and hence the coagulation cascade. Due to its potency, warfarin has become a widely used drug for the prevention of thromboembolic events in patients with heart related conditions like atrial fibrillation and HF with mechanical implants. The therapeutic index of the drug however remains narrow, whiles several other environmental and genetic factors contribute to its efficacy. It is important as a result that patient management on warfarin is individualized to each patients profile. A number of researches have shown that the genetic component of patients using warfarin when combined with age, and body size contributes about 60% in dose variability (10-12). Hence, genetic component plays a significant role in the efficacy of the drug and has even resulted in the creation of a dosing label by the Food and Drugs Administration based on specific genetic polymorphisms as shown in Figure 1. Warfarin exists as a mixture of two stereoisomers: the R-warfarin, and its more functionally effective mirror-molecule, the S-warfarin. Patients with genetic variants in cytochrome P450 complex (CYP2C9*2 or CYP2C9*3) and Vitamin K epoxide reductase complex 1 (VKORC1) have different warfarin metabolism rates that predisposes them to difference sensitivity and drug reactivity (10). CYP2C9 is indicated in the metabolism of the active form of warfarin (S-Warfarin) to its inactive form (7-OH-warfarin). Rare variants of the gene (*2 and *3) however are less effective in metabolizing the drug, increasing the half-life and hence its activity. Sconce et al confirmed this in a study of 297 patients, where they found that warfarin dose, age, body surface area and CYP2C9 significantly contributed to the clearance of the different isoforms of warfarin, especially the S enantiomer (10). The VKORC1 gene on the other hand is responsible for coding the VKORC enzyme, which directly interacts with warfarin. A 1639G>A polymorphism in the promoter region of this gene results in an increase in G allele activity by about 44% compared to the A allele (13). This results in reduced amounts of VKORC1 mRNA and hence less mature VKORC1 enzymes (13). Warfarin dose and management is usually determined or monitored using an International Normalized Ratio (INR), which defines the clotting tendency of the patient’s blood. The INR for a normal individual is between 0.8 and 1.2, with a usual target range of 2.0-3.0 for warfarin therapy. A low INR indicates a high risk of clotting in patients while a higher INR may result in bleeding. Although genotype guided warfarin dosing has been shown to be extensively effective in patients who have atrial fibrillation or non-device related heart problems (12), the role of warfarin genotyping in CF-LVAD patients remains largely unknown.
STUDY OBJECTIVES
The primary goal of this study was to analyze the effect of patient warfarin genotyping on warfarin dose requirements, INR trends, and future thrombotic and bleeding events in patients using CF-LVAD. We hypothesized that warfarin genotyping predicts dosing requirements in CF-LVAD patients, and that warfarin sensitive patients are at higher risk for thrombotic and bleeding complications. We also expected to show that the INR variability of a patient is more telling of an adverse event than the time to therapeutic range. If the INR variability is confirmed to have a higher efficacy, it will reinforce the notion that controlling the INR variability via patient genotype, albeit amidst other possible factors like patient dietary lifestyle, can have a higher impact on possible adverse events.
SPECIFICATION AND VARIABLES
Patients were categorized into two subgroups for the two genotypes, that is CYP2C9 and VKORC1. The subgroup consists of rare variants and wild types. The rare variants will be grouped into clusters, as their frequency is low. Baseline analysis includes number distributions of patient’s ethnicity according to specific CF-LVAD device type, age, gender, CYP2C9 alleles and VKORC1 alleles. Number of patients who are transplanted, or established as long term device therapy patients are also analyzed. Continuous variables like age, warfarin dose and INR information are analyzed as mean and standard deviation. Categorical variables will be analyzed as percentages, with the primary goal of identifying the differences in wild type versus variant genotypes for the various patient characteristics. Statistical methods to be used include independent t-tests for continuous variables and chi-squared test for categorical variables. Log-rank test will be used to test for significance in difference for incidences (GI bleeding, and events). A linear regression analysis will be used to compare difference in warfarin dosing or INR as dependent variable between genotypes, and other factors. Cox proportional analysis can then be used to verify if the genetic variants (CYP2C9 or VKORC1) were independently associated with thrombosis and bleeding. All statistically analysis will be done using R version 3.2.1 platform.
METHODS AND PROCEDURE
There were two parts to this study that included varying methods of data collection and analysis. They included an analysis of both retrospective and prospective data. The retrospective data that was added in this study included patients whose information had been previously collected and analyzed by Topkara et al. These were CF-LVAD patients whose genotype and warfarin information had been recorded between December 2007 and February 2015. New CF-LVAD patient information was added on an ongoing basis with maintenance INR and warfarin dose information of both new and old patients recorded subsequently. The genetic information obtained for the genes of interest and their respective alleles, CYP2C9 (*1, *2 and*3[rs1799853 and rs1057910]) and VKORC1 (-1639G>A [rs9923231]), was done by blood analysis using multiplex PCR, then electrochemical detection with an “e-Sensor” test kit (FDA-cleared assay).
This process was completed at Columbia University Medical Center by the LVAD Team and has culminated in the creation a bio-bank of patient data with information that includes all patient variables as mentioned above in specification and variables. CYP2C9 is considered to have six haplotypes of which patients with *1/*1 are considered to be wild type while those with either *2 or *3 are considered variants. The VKORC gene however has only three haplotypes of which a homozygous G is considered to be the wild type whiles all A haplotypes are considered variants. The first part of the study focused on establishing the genetic associations with warfarin sensitivity among the various ethnic groups and across various factors, and then the next focused on identifying the impact of genome-guided dosing.
All the patients who were recruited for this study had end-stage heart failure followed by a left ventricular assist device implant of one of the following device types: HeartMate II/III, HeartWare/HeartWare BiVAD and Jarvik. Patients who did not qualify for transplant, owing to other complications including another organ failure, were excluded. When notified of a possible candidate, patients were consented by the LVAD team before any sample was obtained. After blood samples were collected and analyzed genetic information was recorded into the iNYP database from where they were retrieved for analysis. The data obtained in this case includes patients age, sex, ethnicity, device type implanted, date of device implant, warfarin dose (after device implant and on discharge), and patient’s maintenance international normalized ratio (INR) which is usually monitored for an optimal range of between 2 -3 mg due to standard warfarin therapy requirement. The time it takes for INR to normalize is also collected.
The dosing regimen for patients in the genotype-guided group was calculated using a website called warfarindosing.org, the same used by the FDA in their warfarin label recommendation, and the aid of a pharmacologist. Outcome data including, gastrointestinal bleeding and epistaxis for all patients were obtained from their medical history. Currently there are 458 patients whose data have been transferred from their medical record into excel.
The current measure of warfarin effectiveness is by the time in therapeutic range (TTR). It has also been confirmed, however, that the variability of the International Normalized Ratio (INR) may indicate thrombotic and bleeding events with a much higher precision. In addition to analyzing the efficacy of genotype-guided warfarin dosing in patients using CF-LVADs, we will also analyze the efficacy of warfarin measurement, using data of CF-LVAD patients from the New York Presbyterian (iNYP) database.
RESULTS AND DISCUSSION
The relationship between warfarin and the international normalized ratio has been evidently established. Patients who have a lower INR usually require a higher warfarin dose, whereas those who whose INR are higher require a lower dose. This interaction allows for the extrapolation of INR analysis and findings with the possible warfarin requirements. The aforementioned study from Topkara et al shows that, patients with both CYP2C9 and VKORC1 variants are sensitive to warfarin and require lower doses. In this study … the
CYP2C9
VKORC1 *1/ *1 *1/ *2 *1/ *3 *2/ *2 *2/ *3 *3/ *3
GG 5-7 mg 5-7 mg 3-4 mg 3-4 mg 3-4 mg 0.5-2 mg
GA 5-7 mg 3-4 mg 3-4 mg 3-4 mg 0.5-2 mg 0.5-2 mg
AA 3-4 mg 3-4 mg 0.5-2 mg 0.5-2 mg 0.5-2 mg 0.5-2 mg
Figure 1: Warfarin dose presentation based on CYP2C9 and VKORC1 Genotypes adapted from the FDA drug label
CONCLUSION
REFERENCES
1. Ni H, Xu JQ. Recent trends in heart failure-related mortality: United States, 2000–2014. NCHS data brief, no 231. Hyattsville, MD: National Center for Health Statistics. 2015.
2. Davis RC, Hobbs FDR, Lip GYH. History and epidemiology. BMJ : British Medical Journal. 320(7226):39-42, 2000
3. Yancy Clyde W., Jessup Mariell, Bozkurt Biykem, et al. 2013 ACCF/AHA Guideline for the Management of Heart Failure: A Report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines Circulation. 128:e240-e327. 2013
4. The Criteria Committee of the New York Heart Association. Nomenclature and Criteria for Diagnosis of Diseases of the Heart and Great Vessels. 9th ed. Boston, Mass: Little, Brown & Co, 253-256. 1994
5. Friedrich EB, Böhm M. Management of end stage heart failure. Heart. 93(5):626-631. doi:10.1136/hrt.2006.098814. 2007
6. Slaughter MS, Rogers JG, Milano CA, et al. Advanced Heart Failure Treated with Continuous-Flow Left Ventricular Assist Device. N Engl J Med 361:2241-51, 2009
7. Crow S, John R, Boyle A, Shumway S, Liao K, Colvin-Adams M, Toninato C, Missov E, Pritzker M, Martin C, Garry D, Thomas W, Joyce L. Gastrointestinal bleeding rates in recipients of nonpulsatile and pulsatile left ventricular assist devices. J Thorac Cardiovasc Surg. Jan;137(1):208-15. 2009
8. Eckman PM, John R. Bleeding and thrombosis in patients with continuous-flow ventricular assist devices. Circulation125(24):3038-47, 2012.
9. Ansell J, Hirsh J, Hylek E, Jacobson A, Crowther M, Palareti G. “Pharmacology and management of the vitamin K antagonists: American College of Chest Physicians evidence-based clinical practice guidelines (8th Edition)” Chest. 133 (6 Suppl): 160S–198S.
10. Sconce EA, Khan T. Wayne HA, Avery P, Monkhouse L, King BP, Wood P, Kesteven P, Daly AK, Kamali F. The impact of CYP2C9 and VKORC1 genetic polymorphism and patient characteristics upon warfarin dose requirements: proposal for a new dosing regimen. The American Society of Hematology. 2005:2329-2333
11. Vecsler, M, Loebstein, R, Almog, S, et al Combined genetic profiles of components and regulators of the vitamin K-dependent gamma-carboxylation system affect individual sensitivity to warfarin.Thromb Haemost2006;95,205-211
12. Herman, D, Peternel, P, Stegnar, M, et al The influence of sequence variations in factor VII, gamma-glutamyl carboxylase and vitamin K epoxide reductase complex genes on warfarin dose requirement.Thromb Haemost2006;95,782-787
13. Owen RP, Gong L, Sagreiya H, Klein TE, Altman RB. VKORC1 Pharmacogenomics Summary. Pharmacogenetics and Genomics. 2010;20(10):642-644. doi:10.1097/FPC.0b013e32833433b6.
10. Topkara VK, Knotts RJ, Garan AR et al. Effect of CYP2C9 and VKORC1 Gene Variants on Warfarin Response and Clinical Events in Patients with Continuous-Flow Left Ventricular Assist Devices (CF-LVADs). ASAIO. In Press.
11. Gage BF, Lesko LJ. Pharmacogenetics of warfarin: regulatory, scientific, and clinical issues.. J Thromb Thrombolysis. Feb;25(1):45-51. 2008
12. Dean L. Warfarin Therapy and the Genotypes CYP2C9 and VKORC1. 2012 Mar 8 [Updated 2013 Mar 18]. In: Medical Genetics Summaries [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2012
13. Pirmohamed M, Burnside G, et al A randomized Trial of Genotype-Guided Dosing of Warfarin. N Engl J Med 2013;3692294-2303