Multiplex assay for comprehensive genotyping of genes involved in drug metabolism, excretion, and transport.

BACKGROUND: Drug metabolism is a multistep process by which the body disposes of xenobiotic agents such as therapeutic drugs. Genetic variation in the enzymes involved in this process can lead to variability in a patient's response to medication. METHODS: We used molecular-inversion probe technology to develop a multiplex genotyping assay that can simultaneously test for 1227 genetic variants in 169 genes involved in drug metabolism, excretion, and transport. Within this larger set of variants, we performed analytical validation of a clinically defined core set of 165 variants in 27 genes to assess accuracy, imprecision, and dynamic range. RESULTS: In a test set of 91 samples, genotyping accuracy for the core set probes was 99.8% for called genotypes, with a 1.2% no-call (NC) rate. The majority of the core set probes (133 of 165) had < or = 1 genotyping failure in the test set; a subset of 12 probes was responsible for the majority of failures (mainly NC). Genotyping results were reproducible upon repeat testing with overall within- and between-run variation of 1.1% and 1.4%, respectively-again, primarily NCs in a subset of probes. The assay showed stable genotyping results over a 6-fold range of input DNA. CONCLUSIONS: This assay generates a comprehensive assessment of a patient's metabolic genotype and is a tool that can provide a more thorough understanding of patient-to-patient variability in pharmacokinetic responses to drugs.

Comprehensive assessment of metabolic enzyme and transporter genes using the Affymetrix Targeted Genotyping System.

The combined effects of multiple polymorphisms in several drug-metabolizing enzyme and transporter genes can contribute to considerable interindividual variation in drug disposition and response. Therefore, it has been of increasing interest to generate scalable, flexible and cost-effective technologies for large-scale genotyping of the drug-metabolizing enzyme and transporter genes. However, the number of drug-metabolizing enzyme and transporter gene variants exceeds the capacity of current technologies to comprehensively assess multiple polymorphisms in a single, multiplexed assay. The Targeted Genotyping System (Affymetrix, CA, USA) provides a solution to this challenge, by combining molecular inversion probe technology with universal microarrays to provide a method that is capable of analyzing thousands of variants in a single reaction, while remaining relatively insensitive to cross-reactivity between reaction components. This review will focus on the Targeted Genotyping System and how this technology was adapted to enable comprehensive analysis of drug-metabolizing enzyme and transporter gene polymorphisms.

A phase II trial of pemetrexed in advanced breast cancer: clinical response and association with molecular target expression.

PURPOSE: This phase II trial of pemetrexed explored potential correlations between treatment outcome (antitumor activity) and molecular target expression. EXPERIMENTAL DESIGN: Chemonaïve patients with advanced breast cancer received up to three cycles of pemetrexed 500 mg/m2 (10-minute i.v. infusion) on day 1 of a 21-day cycle, with folic acid and vitamin B12 supplementation. Tumors were surgically removed after the last cycle of pemetrexed as clinically indicated. Biopsies were taken at baseline, 24 hours after infusion in cycle 1, and after cycle 3. RESULTS: Sixty-one women (median age, 46 years; range, 32-72 years) were treated and were evaluable for response. Objective response rate was 31%. Simple logistic regression suggested a potential relationship between mRNA expression of thymidylate synthase (TS) and pemetrexed response (P = 0.103). Based on threshold analysis, patients with "low" baseline TS (< or = 71) were more likely to respond to pemetrexed than patients with "high" baseline TS (>71). Expression of baseline dihydrofolate reductase and glycinamide ribonucleotide formyl transferase tended to be higher in responders but this association was not significant (P > 0.311). TS expression increased significantly between baseline and biopsy 2 (P = 0.004) and dropped to near baseline levels at biopsy 3. Conversely, dihydrofolate reductase and glycinamide ribonucleotide formyl transferase decreased after pemetrexed chemotherapy. CONCLUSIONS: Our results suggest a potential association between "low" pretreatment TS expression levels and response to pemetrexed chemotherapy. Future trials examining expression levels of other genes important to the folate pathway and/or breast cancer may identify a more robust multigene profile that can better predict response to this novel antifolate.

Home management of sickle cell-related pain in children and adolescents: natural history and impact on school attendance.

Some children and adolescents with sickle cell disease experience frequent painful episodes. To gain information about the natural history of the pain and its impact on sleep and school attendance, we developed a home-based diary system. Eighteen children and adolescents completed 4756 diary days, with an average compliance of 75%. Pain was reported on 30% of days and was managed at home nine-tenths of the time. Girls reported more days with pain than did boys, and age was positively correlated with the length of the painful episodes. The pain affected school attendance and sleep. Patients were absent from school on 21% of 3186 school days, with half of the absenteeisms on days with reported pain. Of the pain-associated absenteeisms, two-thirds occurred when pain was managed at home, and one-third when patients were hospitalized. The average consecutive number of school days missed was 2.7. These findings have implications for developmentally critical activities.