DNA isolated from formalin fixed paraffin embedded (FFPE) tissue is often fragmented and cross linked and is therefore difficult to genotype To enable this source of DNA for genotyping analysis... Show moreDNA isolated from formalin fixed paraffin embedded (FFPE) tissue is often fragmented and cross linked and is therefore difficult to genotype To enable this source of DNA for genotyping analysis using Taqman probes, we tested whether enrichment of the target genes would increase the amount of available DNA For enrichment of the target genes, we used preamplification by means of diluted Taqman assays To establish the appropriateness of preamplification, we used DNA extracted from paraffin embedded tissue and compared the genotyping results of a series of single nucleotide polymorphisms assessed m DNA samples with and without preamplification In a subset of patients, DNA was isolated from both blood and FFPE tissue to test the reliability of genotyping results derived after preamplification We found an increase in call rate after preamplification and a convincing con cordance in genotype Based on our findings, we can safely conclude that preamplification of DNA isolated from paraffin-embedded tissue is a valuable and reliable method to optimize genotyping results (J Mol Diagn 2010, 12 746-749, DOI 10.2353/jmoldx.2010.100047) Show less
Pander, J.; Wessels, J.A.M.; Gelderblom, H.; Straaten, T. van der; Punt, C.J.A.; Guchelaar, H.J. 2010
BACKGROUND: Pharmacogenetic markers related to drug metabolism and mechanisms of action could help to better select patients with metastatic colorectal cancer (mCRC) for treatment. Genetic... Show moreBACKGROUND: Pharmacogenetic markers related to drug metabolism and mechanisms of action could help to better select patients with metastatic colorectal cancer (mCRC) for treatment. Genetic interaction analysis is used as a rational tool to study the contribution of polygenic variation in relation to drug response. PATIENTS AND METHODS: A selection of 17 polymorphisms in genes encoding drug targets, pathway molecules and detoxification enzymes was analyzed in 279 previously untreated mCRC patients treated with capecitabine, oxaliplatin and bevacizumab (CAPOX-B). Multifactor dimensionality reduction analysis was used to identify a genetic interaction profile for progression-free survival (PFS). RESULTS: Median PFS was 10.9 [95% confidence interval (CI) 9.4-12.4] months. A genetic interaction profile consisting of the TYMS enhancer region and VEGF +405G>C polymorphisms was significantly associated with PFS. Median PFS was 13.3 (95% CI 11.4-15.3) and 9.7 (95% CI 7.6-11.8) months for the beneficial and unfavorable genetic profiles, respectively, corresponding to a hazards ratio for PFS of 1.58 (95% CI 1.14-2.19). None of the studied polymorphisms were individually associated with PFS. CONCLUSIONS: Our results support a genetic interaction between the TYMS enhancer region and VEGF +405G>C polymorphisms as a predictor of the efficacy of CAPOX-B in mCRC patients. Show less
Pander, J.; Gelderblom, H.; Antonini, N.F.; Tol, J.; Krieken, J.H.J.M. van; Straaten, T. van der; ... ; Guchelaar, H.J. 2010
Background: Next to KRAS mutation status, additional predictive markers are needed for the response to cetuximab in patients with metastatic colorectal cancer (mCRC). Previous studies indicated... Show moreBackground: Next to KRAS mutation status, additional predictive markers are needed for the response to cetuximab in patients with metastatic colorectal cancer (mCRC). Previous studies indicated that germline polymorphisms in specific genes may predict efficacy and toxicity of cetuximab in mCRC patients. Methods: Germline DNA was isolated from 246 KRAS wild-type mCRC patients who were treated in the phase III CAIRO2 study with chemotherapy and bevacizumab alone or with the addition of cetuximab. Associations of epidermal growth factor (EGF) 61A > G, EGF receptor (EGFR) CA(14-22), cyclin D1 (CCND1) 932G > A, fragment-C gamma receptor (FCGR) 2A 535A > G and FCGR3A 818A > C polymorphisms with progression-free survival (PFS) and cetuximab-related skin toxicity were studied. Results: In cetuximab-treated patients, the FCGR3A 818C-allele was associated with decreased PFS compared with the FCGR3A 818AA genotype (median PFS, 8.2 [95%CI, 6.7-10.3] versus 12.8 [95%CI, 10.3-14.7] months, respectively; HR, 1.57 [95%CI, 1.06-2.34]; P = .025). The EGFR 20 genotype was associated with decreased PFS compared with the EGFR < 20 genotype (median PFS, 7.6 [95%CI, 6.7-10.0] versus 12.4 [95%CI, 10.3-13.4] months, respectively; HR, 1.58 [95%CI, 1.06-2.35]; P = .024). The FCGR3A and EGFR polymorphisms were not associated with PFS in patients treated without cetuximab. None of the polymorphisms were associated with the incidence of grades 2-3 skin toxicity. Conclusion: EGFR and FCGR3A germline polymorphisms are associated with PFS in KRAS wild-type mCRC patients treated with cetuximab, bevacizumab and chemotherapy. (C) 2010 Elsevier Ltd. All rights reserved. Show less
Press, R.R.; Ploeger, B.A.; Hartigh, J. den; Straaten, T. van der; Pelt, H. van; Danhof, M.; ... ; Guchelaar, H.J. 2010
Optimal ciclosporin A (CsA) exposure in kidney transplant recipients is difficult to attain because of variability in CsA pharmacokinetics. A better understanding of the variability in CsA exposure... Show moreOptimal ciclosporin A (CsA) exposure in kidney transplant recipients is difficult to attain because of variability in CsA pharmacokinetics. A better understanding of the variability in CsA exposure could be a good means of individualizing therapy. Specifically, genetic variability in genes involved in CsA metabolism could explain exposure differences. Therefore, this study is aimed at identifying a relationship between genetic polymorphisms and the variability in CsA exposure, while accounting for non-genetic sources of variability. De novo kidney transplant patients (n = 33) were treated with CsA for 1 year and extensive blood sampling was performed on multiple occasions throughout the year. The effects of the non-genetic covariates hematocrit, serum albumin concentration, cholesterol, demographics (i.e., body weight), CsA dose interval, prednisolone dose and genetic polymorphisms in genes encoding ABCB1, CYP3A4, CYP3A5, and PXR on CsA pharmacokinetics were studied using non-linear mixed effect modeling. The pharmacokinetics of CsA were described by a two-compartment disposition model with delayed absorption. Body weight was identified as the most important covariate and explained 35% of the random inter-individual variability in CsA clearance. Moreover, concurrent prednisolone use at a dosage of 20 mg/day or higher was associated with a 22% higher clearance of CsA, hence lower CsA exposure. In contrast, no considerable genotype effects (i.e., greater than 30-50%) on CsA clearance were found for the selected genes. It appears that the selected genetic markers explain variability in CsA exposure insufficiently to be of clinical relevance. Therefore, therapeutic drug monitoring is still required to optimize CsA exposure after administration of individualized doses based on body weight and, as this study suggests, co-administration of prednisolone. Show less
Pander, J.; Gelderblom, H.; Straaten, T. van der; Punt, C.J.A.; Guchelaar, H.J. 2010
Aims: Clinical response to methotrexate (MTX) treatment differs among rheumatoid arthritis patients. Genetic variation can partly account for this phenomenon. In this study, functional... Show moreAims: Clinical response to methotrexate (MTX) treatment differs among rheumatoid arthritis patients. Genetic variation can partly account for this phenomenon. In this study, functional polymorphisms in genes related to the mechanism of action of MTX or immunopathogenesis of rheumatoid arthritis were studied for association with treatment outcome in a Dutch cohort of patients with early rheumatoid arthritis. Furthermore, tests for replication of previous research on these genetic variants were performed according to reported end points. Materials & methods: Seven polymorphisms in seven genes were analyzed in 205 genotyped patients with active rheumatoid arthritis. All patients received standardized MTX treatment (<= 25 mg per week orally) combined with folic acid. MTX treatment outcome was evaluated by disease activity score criteria and adverse drug events. The following genetic variants were analyzed and correlated: ABCB1 3435C > T, ITPA IVS2 +21A > C, HLA-G (-14 bp >+14 bp), TGFB1 +869T > C and TLR4 +896A > G. In case of significant differences, regression analyses were applied. Since carriers of the minor alleles of the SNPs DHFR 829C > T and IMPDH2 +787C > T were not observed, no statistical analyses could be performed. Results: No significant associations or replications of these genetic variants with MTX efficacy were demonstrated. Regarding toxicity, patients carrying the ABCB1 3435T-allele and TLR4 +896G-allele were 2.5-times more likely to develop adverse drug events at 6 months (odds ratio: 2.6; 95% CI: 1.1-6.2, and odds ratio: 2.5; 95% CI: 1.1-6.1, respectively). Additionally, this chance increased almost fourfold in patients with the two unfavorable genotypes (odds ratio: 3.9; 95% CI: 1.5-10.3). However, none of these associations remained significant after correction for multiple testing (p < 0.004). Conclusion: Our data indicate that MTX toxicity was potentially associated with ABCB1 3435C > T and TLR4 +896A > G. However, after correction, none of these associations remained significant. Furthermore, no significant associations or replications of these functional variants with efficacy were found. Show less
With the exponential increase in publications on DNA markers explaining and/or predicting response to drug therapy, the potential of pharmacogenetic testing of individual patients to optimize drug... Show moreWith the exponential increase in publications on DNA markers explaining and/or predicting response to drug therapy, the potential of pharmacogenetic testing of individual patients to optimize drug treatment is expanding. For the identification of pharmacogenetic markers, several techniques can be used. The specific method usually depends on the requirements of the study, ranging from determining one or two single nucleotide polymorphisms (SNPs) in one to ten patients, one or two SNPs in thousands of patients, several thousands of SNPs in an individual patient, or thousands of SNPs in thousands of patients. In this review we identify and evaluate the information present in the literature on genotyping assays that are currently used for pharmacogenetic analyses, and discuss the advantages and disadvantages of these techniques. Show less
