Dennis J. O'Kane

Warfarin Genotyping Reduces Hospitalization Rates Results From the MM-WES (Medco-Mayo Warfarin Effectiveness Study)

OBJECTIVES This study was designed to determine whether genotype testing for patients initiating warfarin treatment will reduce the incidence of hospitalizations, including those due to bleeding or thromboembolism. BACKGROUND Genotypic variations in CYP2C9 and VKORC1 have been shown to predict warfarin dosing, but no large-scale studies have prospectively evaluated the clinical effectiveness of genotyping in naturalistic settings across the U.S. METHODS This national, prospective, comparative effectiveness study compared the 6-month incidence of hospitalization in patients receiving warfarin genotyping (n = 896) versus a matched historical control group (n = 2,688). To evaluate for temporal changes in the outcomes of warfarin treatment, a secondary analysis compared outcomes for 2 external control groups drawn from the same 2 time periods. RESULTS Compared with the historical control group, the genotyped cohort had 31% fewer hospitalizations overall (adjusted hazard ratio [HR]: 0.69, 95% confidence interval [CI]: 0.58 to 0.82, p < 0.001) and 28% fewer hospitalizations for bleeding or thromboembolism (HR: 0.72, 95% CI: 0.53 to 0.97, p = 0.029) during the 6-month follow-up period. Findings from a per-protocol analysis were even stronger: 33% lower risk of all-cause hospitalization (HR: 0.67, 95% CI: 0.55 to 0.81, p < 0.001) and 43% lower risk of hospitalization for bleeding or thromboembolism (HR: 0.57, 95% CI: 0.39 to 0.83, p = 0.003) in patients who were genotyped. During the same period, there was no difference in outcomes between the 2 external control groups. CONCLUSIONS Warfarin genotyping reduced the risk of hospitalization in outpatients initiating warfarin. (The Clinical and Economic Impact of Pharmacogenomic Testing of Warfarin Therapy in Typical Community Practice Settings [MHSMayoWarf1]; NCT00830570).

COMPARISON OF SCREENING METHODS IN THE DETECTION OF BLADDER CANCER

PURPOSE We prospectively evaluate and compare the sensitivity and specificity of urine cytology, BTA stat, NMP22, fibrin/fibrinogen degradation products (FDP), telomerase, chemiluminescent hemoglobin and hemoglobin dipstick to detect bladder cancer. MATERIALS AND METHODS Single voided specimens were obtained from 57 patients with bladder cancer, and 139 without evidence of bladder malignancy on cystoscopy or a negative biopsy of indeterminate lesions. A cytology report was available for 125 patients and interpreted independently. BTA stat, NMP22 and FDP were analyzed according to manufacturer specifications. The telomerase assay was performed on cells collected from urine by centrifugation in preparation for polymerase chain reaction based amplification using the telomeric repeat amplification protocol assay. The chemiluminescent screening assay for hemoglobin in urine uses the pseudoperoxidase activity of hemoglobin on hydrogen peroxide and subsequent oxidation of 7-dimethylaminonaphthalene-1,2-dicarbonic acid hydrazide to generate chemiluminescence emission. Hemoglobin dipstick was interpreted as positive if the hemoglobin content in the urine was trace or greater. RESULTS Overall sensitivity with urine cytology, BTA stat, NMP22, FDP, telomerase, chemiluminescent hemoglobin and the hemoglobin dipstick was 44, 74, 53, 52, 70, 67 and 47%, respectively. Specificity with cytology, telomerase and FDP was high (95, 99 and 91%, respectively) but BTA stat, NMP22 (optimized), chemiluminescent hemoglobin (optimized) and the hemoglobin dipstick demonstrated lower specificity of 73, 60, 63 and 84%, respectively. Stepwise logistic regression analysis revealed that for all tumors, and within each tumor grade and stage telomerase had the strongest association with bladder cancer among all tests (69% overall concordance). Telomerase was also positive in 91% of the patients (10 of 11) with carcinoma in situ. CONCLUSIONS Urinary telomerase had the highest combination of sensitivity and specificity (70 and 99%, respectively) for bladder cancer screening in these patients. It was the strongest predictor with superior accuracy in patients with grade 1 and noninvasive tumors (pTa), and extremely useful in patients with carcinoma in situ. Telomerase appears to be promising and outperformed cytology, BTA stat, NMP22, FDP, chemiluminescent hemoglobin and hemoglobin dipstick in the prediction of bladder cancer.

Pharmacogenetic testing of CYP2C9 and VKORC1 alleles for warfarin

Disclaimer: American College of Medical Genetics statements and guidelines are designed primarily as an educational resource for medical geneticists and other health care professionals to help them provide quality medical genetic services. Adherence to these standards and guidelines does not necessarily ensure a successful medical outcome. These statements and guidelines should not be considered inclusive of all proper procedures and tests or exclusive of other procedures and tests that are reasonably directed to obtaining the same results. In determining the propriety of any specific procedure or test, the health care professional should apply his or her own professional judgment to the specific clinical circumstances presented by the individual patient or specimen. It may be prudent, however, to document in the patients record the rationale for any significant deviation from these standards and guidelines.Warfarin (Coumadin) is a potent drug that when used judiciously and monitored closely, leads to substantial reductions in morbidity and mortality from thromboembolic events. However, even with careful monitoring, initiation of warfarin dosing is associated with highly variable responses between individuals and challenges achieving and maintaining levels within the narrow therapeutic range that can lead to adverse drug events. Variants of two genes, CYP2C9 and VKORC1, account for 30–50% of the variability in dosing of warfarin; thus, many believe that testing of these genes will aid in warfarin dosing recommendations. Evidence about this test is evolving rapidly, as is its translation into clinical practice. In an effort to address this situation, a multidisciplinary expert group was organized in November 2006 to evaluate the role of CYP2C9 and VKORC1 testing in altering warfarin-related therapeutic goals and reduction of adverse drug events. A recently completed Rapid-ACCE (Analytical, Clinical Validity, Clinical Utility, and Ethical, Legal, and Social Implications) Review, commissioned to inform this work group, was the foundation for this analysis. From this effort, specific recommendations for the appropriate use of CYP2C9 and VKORC1 testing were developed and are presented here. The group determined that the analytical validity of these tests has been met, and there is strong evidence to support association between these genetic variants and therapeutic dose of warfarin. However, there is insufficient evidence, at this time, to recommend for or against routine CYP2C9 and VKORC1 testing in warfarin-naive patients. Prospective clinical trials are needed that provide direct evidence of the benefits, disadvantages, and costs associated with this testing in the setting of initial warfarin dosing. Although the routine use of warfarin genotyping is not endorsed by this work group at this time, in certain situations, CYP2C9 and VKORC1 testing may be useful, and warranted, in determining the cause of unusual therapeutic responses to warfarin therapy.

Non-invasive in vivo monitoring of trackable viruses expressing soluble marker peptides.

Noninvasive methods are needed to study the kinetic properties of viruses in living organisms. Oncolytic viruses are used increasingly for cancer therapy but there is currently no satisfactory way to measure efficiency of tumor transduction, changing levels of viral gene expression or the timing of virus elimination. We therefore generated trackable oncolytic measles viruses expressing inert (nonimmunogenic, nonfunctional and accurately measurable) soluble marker peptides. The marker peptides did not compromise virus replication. Ex vivo and in vivo kinetics of the trackable viruses could be easily followed by measuring the concentrations of virally encoded marker peptides in culture supernatant or in serum. When mice bearing human tumor xenografts were challenged with the trackable viruses, distinct kinetic profiles of marker-gene expression could be correlated with distinct therapeutic outcomes. Oncolytic viruses expressing inert soluble marker polypeptides should greatly facilitate the rational development of effective, individually tailored cancer virotherapy.

SLC6A4 variation and citalopram response.

The influence of genetic variations in SLC6A4 (serotonin transporter gene) on citalopram treatment of depression using the Sequenced Treatment to Relieve Depression (STAR*D) sample was assessed. Of primary interest were three previously studied polymorphisms: 1) the VNTR variation of the second intron, 2) the indel promoter polymorphism (5HTTLPR or SERT), and 3) a single nucleotide polymorphism (SNP) rs25531. Additionally, SLC6A4 was resequenced to identify new SNPs for exploratory analyses. DNA from 1914 subjects in the STAR*D study were genotyped for the intron 2 VNTR region, the indel promoter polymorphism, and rs25531. Associations of these variants with remission of depressive symptoms were evaluated following citalopram treatment. In white non‐Hispanic subjects, variations in the intron 2 VNTR (point‐wise P = 0.041) and the indel promoter polymorphism (point‐wise P = 0.039) were associated with remission following treatment with citalopram. The haplotype composed of the three candidate loci was also associated with remission, with a global p‐value of 0.040 and a maximum statistic simulation p‐value of 0.0031 for the S‐a‐12 haplotype, under a dominant model. One SNP identified through re‐sequencing the SLC6A4 gene, Intron7‐83‐TC, showed point‐wise evidence of association, which did not remain significant after correction for the number of SNPs evaluated in this exploratory analysis. No associations between these SLC6A4 variations and remission were found in the white Hispanic or black subjects. These findings suggest that multiple variations in the SLC6A4 gene are associated with remission in white non‐Hispanic depressed adults treated with citalopram. The mechanism of action of these variants remains to be determined.

DIAGNOSTIC PERFORMANCE OF CYST FLUID CARCINOEMBRYONIC ANTIGEN AND AMYLASE IN HISTOLOGICALLY CONFIRMED PANCREATIC CYSTS

Objectives: The objective of this study was to evaluate and validate cyst fluid carcinoembyronic antigen (CEA) and amylase in differentiating (1) nonmucinous from mucinous pancreatic cystic lesions (PCLs), (2) benign mucinous from malignant mucinous PCLs, and (3) pseudocysts from nonpseudocysts (amylase only). Methods: A retrospective analysis of patients with histologically confirmed PCLs from February 1996 to April 2007 was performed. Cyst fluid CEA (n = 124) and/or amylase (n = 91) were measured and correlated to cyst type. Results: Carcinoembyronic antigen levels (P = 0.0001), but not amylase, were higher in mucinous versus nonmucinous cysts. The sensitivity, specificity, and diagnostic accuracy of CEA 200 ng/mL or greater for the diagnosis of mucinous PCLs were 60%, 93%, and 72%, respectively. Carcinoembyronic antigen levels did not differentiate benign from malignant mucinous cysts. Whereas amylase levels were higher in pseudocysts than nonpseudocysts (P = 0.009), 54% of noninflammatory PCLs had a level greater than 250 IU/L, including mucinous cystic neoplasms (median, 6800 IU/L; interquartile range, 70-25,295 IU/L). Malignant mucinous cysts had lower amylase levels than benign mucinous cysts (P = 0.0008). Conclusions: Cyst fluid CEA and amylase levels are suggestive but not diagnostic in differentiating PCLs. Unlike CEA, amylase may help differentiate benign from malignant mucinous cysts. Novel biomarkers are needed.

Warfarin Sensitivity Genotyping: A Review of the Literature and Summary of Patient Experience

The antithrombotic benefits of warfarin are countered by a narrow therapeutic index that contributes to excessive bleeding or cerebrovascular clotting and stroke in some patients. This article reviews the current literature describing warfarin sensitivity genotyping and compares the results of that review to the findings of our study in 189 patients at Mayo Clinic conducted between June 2001 and April 2003. For the review of the literature, we identified relevant peer-reviewed articles by searching the Web of Knowledge using key word warfarin-related adverse event. For the 189 Mayo Clinic patients initiating warfarin therapy to achieve a target international normalized ratio (INR) in the range of 2.0 to 3.5, we analyzed the CYP2C9 (cytochrome P450 2C9) and VKORC1 (vitamin K epoxide reductase complex, subunit 1) genetic loci to study the relationship among the initial warfarin dose, steady-state dose, time to achieve steady-state dose, variations in INR, and allelic variance. Results were compared with those previously reported in the literature for 637 patients. The relationships between allelic variants and warfarin sensitivity found in our study of Mayo Clinic patients are fundamentally the same as in those reported by others. The Mayo Clinic population is predominantly white and shows considerable allelic variability in CYP2C9 and VKORC1. Certain of these alleles are associated with increased sensitivity to warfarin. Polymorphisms in CYP2C9 and VKORC1 have a considerable effect on warfarin dose in white people. A correlation between steady-state warfarin dose and allelic variants of CYP2C9 and VKORC1 has been demonstrated by many previous reports and is reconfirmed in this report. The allelic variants found to most affect warfarin sensitivity are CYP2C9*1*1-VKORC1BB (less warfarin sensitivity than typical); CYP2C9*1*1-VKORC1AA (considerable variance in INR throughout initiation); CYP2C9*1*2-VKORC1AB (more sensitivity to warfarin than typical); CYP2C9*1*3-VKORC1AB (much more sensitivity to warfarin than typical); CYP2C9*1*2-VKORC1AB (much more sensitivity to warfarin than typical); CYP2C9*1*3-VKORC1AA (much more sensitivity to warfarin than typical); and CYP2C9*2*2-VKORC1AB (much more sensitivity to warfarin than typical). Although we were unable to show an association between allelic variants and initial warfarin dose or dose escalation, an association was seen between allelic variant and steady-state warfarin dose. White people show considerable variance in CYP2C9 allele types, whereas people of Asian or African descent infrequently carry CYP2C9 allelic variants. The VKORC1AA allele associated with high warfarin sensitivity predominates in those of Asian descent, whereas white people and those of African descent show diversity, carrying either the VKORC1BB, an allele associated with low warfarin sensitivity, or VKORC1AB or VKORC1AA, alleles associated with moderate and high warfarin sensitivity, respectively.

Endothelial nitric oxide synthase gene polymorphisms predict susceptibility to aneurysmal subarachnoid hemorrhage and cerebral vasospasm

Rupture of an intracranial aneurysm (subarachnoid hemorrhage) is a potentially devastating condition frequently complicated by delayed cerebral ischemia from sustained contraction of intracranial arteries (cerebral vasospasm). There is mounting evidence linking the formation of intracranial aneurysms and the pathogenesis of post-subarachnoid hemorrhage vasospasm to aberrant bioavailability and action of the vasodilator molecule nitric oxide generated by isoforms of nitric oxide synthase. In humans, the gene encoding the endothelial isoform of nitric oxide synthase (eNOS) is known to be polymorphic, with certain polymorphisms associated with increased cardiovascular disease susceptibility. In this prospective clinical study involving 141 participants, we used gene microarray technology to demonstrate that the eNOS gene intron-4 27-base pair variable number tandem repeat polymorphism (eNOS 27 VNTR) predicts susceptibility to intracranial aneurysm rupture, while the eNOS gene promoter T-786C single nucleotide polymorphism (eNOS T-786C SNP) predicts susceptibility to post-subarachnoid hemorrhage vasospasm. We believe that genetic information such as this, which can be obtained expeditiously at the time of diagnosis, may be used as a helpful adjunct to other clinical information aimed at predicting and favorably modifying the clinical course of persons with intracranial aneurysms.

CYP2C19 Variation and Citalopram Response

Objective Variations in cytochrome P450 (CYP) genes have been shown to be associated with both accelerated and delayed pharmacokinetic clearance of many psychotropic medications. Citalopram is metabolized by three CYP enzymes. CYP2C19 and CYP3A4 play a primary role in citalopram metabolism, whereas CYP2D6 plays a secondary role. Methods The Sequenced Treatment Alternatives to Relieve Depression sample was used to examine the relationship between variations in the CYP2C19 and CYP2D6 genes and remission of depressive symptoms and tolerance to treatment with citalopram. The primary analyses were of the White non-Hispanic patients adherent to the study protocol (n=1074). Results Generally, patients who had CYP2C19 genotypes associated with decreased metabolism were less likely to tolerate citalopram than those with increased metabolism, although this difference was not statistically significant (P=0.06). However, patients with the inactive 2C19*2 allele had significantly lower odds of tolerance (P=0.02). Patients with the poor metabolism CYP2C19 genotype-based category who were classified as citalopram tolerant were more likely to experience remission (P=0.03). No relationship between CYP2D6 genotype-based categories and either remission or tolerance was identified, although exploratory analyses identified a potential interaction between CYP2C19 and CYP2D6 effects. Conclusion Despite several limitations including the lack of serum drug levels, this study showed that variations in CYP2C19 were associated with tolerance and remission in a large sample of White non-Hispanic patients treated with citalopram.

Endothelial Nitric Oxide Synthase T-786C Single Nucleotide Polymorphism A Putative Genetic Marker Differentiating Small Versus Large Ruptured Intracranial Aneurysms

Background and Purpose— Anecdotal evidence exists for at least 2 subpopulations of intracranial saccular aneurysms, namely, those that may form rapidly and rupture when small versus those that enlarge slowly and may rupture particularly when ≥10 mm in diameter. We sought to determine whether the endothelial nitric oxide synthase (eNOS) T-786C single nucleotide polymorphism (SNP), implicated in cardiovascular disease susceptibility, could facilitate differentiation between small (≤5 mm) versus large (≥10 mm) ruptured aneurysms. Methods— In accordance with institutional guidelines, clinical data were recorded prospectively and genomic DNA was isolated from blood samples obtained from 52 aneurysmal subarachnoid hemorrhage (SAH) patients (cases) and 90 randomly selected controls. Samples were assayed for eNOS gene promoter T-786C SNP with the use of gene microarray technology. Statistical analyses included multiple logistic regression. Results— Although there was no difference in genotype distributions between cases and controls, all 13 patients with large aneurysms were (T/ C) heterozygous for the polymorphism, while 9 of 22 patients (41%) with small aneurysms were (T/ T or C/ C) homozygous (P =0.01). The mean (±SD) ruptured aneurysm diameter among all heterozygotes (8.5±5.2 mm) was significantly greater than that for C/ C (6.0±2.3 mm) or T/ T (4.7±1.8 mm) homozygotes (P =0.04). With the use of multivariate analysis, heterozygosity remained significantly associated with aneurysm size ≥10 mm (P =0.03). Conclusions— The eNOS T-786C SNP distinguishes genetically between small and large ruptured aneurysms. Although not predictive of SAH in the population at large, our data suggest that among persons with known intracranial aneurysms, eNOS T-786C genotype may be a factor influencing the size at which an aneurysm ruptures, a finding that should be taken into consideration along with other anatomic features of the aneurysm.