Sub theme 1.5

Goals of research: general outline
Scientific achievements
Future plans: special goals and approach
Running projects
Associated staff

Workgroup leaders   Department
Prof.dr.  J.  Lindemans   Clinical Chemistry
Dr.  R.  van  Schaik   Internal Medicine
Prof.Dr  A.G.  Uitterlinden   Internal Medicine

Goals of research: general outline

The focus of this program is on molecular markers in body fluids that predict the effectiveness and toxicities of therapeutic interventions. In particular, the contribution of genetic predisposition of enzymes involved in the metabolism of endogenous and exogenous substrates is investigated. Part of this research programm is focussed on endogenous methylation and methyl-group  metabolism in relation to vascular disease and oncology (project leaders Prof. Dr. Jan Lindemans and Dr. Robert de Jonge).

The translational research performed is aimed to identify and evaluate the clinical significance of genetic polymorphisms (SNPs/haplotypes) in relation to pharmacokinetic and pharmacodynamics of medicines. Targets are genes encoding drug metabolizing enzymes (a.o. cytochrome P450s, UGTs) and drug transporters (a.o. MDR1, MRP2, etc). Ultimate aim is to develop and incorporate clinically significant pharmacogenetic tests for routine patient care.

Specific research topics include pharmacogenetics of folate antimetabolites (methotrexate, pemetrexed, collaboration with Depts. Rheumatology, Neurology and Pulmonology), immunosuppressives (collaboration with Dept. Hospital Pharmacy), drugs in oncology (tamoxifen and taxanes, collaboration with Dept. Medical Oncology), anti-depressants and anti-epileptics (collaboration with Dept. Psychiatry), anaesthetics (collaboration with Dept. Anesthesiology), anti-virals (collaboration with Dept. Internal Medicine), anti-coagulation drugs (collaboration with Dept. Epidemiology & Biostatistics) and opioids (collaboration with Depts. Children’s Surgery and Medical Oncology).

Scientific achievements

The department has actively introduced and stimulated pharmacogenetic research into a number of clinical area’s, involving immunosuppressive drugs, antivirals, anti-cancer drugs, antidepressants and antipsychotics, pain medication, rheumatolology and anticoagulation drugs.

One of the first contributions was the discovery of the high prevalence of CYP3A5 polymorphisms in the Dutch population, following development of assays for these genetic polymorphisms. In a collaboration with Dpet. Internal Medicine and Hospital Pharmacy, we were one of the first groups to demonstrate the clinical importance of CYP3A5 polymorphisms in immunosuppressive therapy with tacrolimus, resulting in a two-fold lower exposure to this drug in the 20% CYP3A5 expressers in the Caucasian population. Subsequent further studies also clarified the role of CYP3A4 and MDR-1 polymorphisms for both tacrolimus and cyclosporin therapy. Recently, we were the first group worldwide to identify UGT1A9 polymorphims as being related not only to mycophenolate mofetyl pharmacokinetics, a drug commonly used in solid organ transplantations, but also to the clinical endpoint of acute kidney rejections. The through these projects assembled biobank at our department of over 340 kidney transplant patients serves as a valuable tool to further investigate the role of pharmacogenetics in relation to immunosuppressive therapy.

In collaboration with the Dept. Epidemiology & Biostatistics, we screened the entire ERGO population for genetic polymorphisms in drug metabolizing enzymes CYP2C19, CYP2C9, CYP2D6, CYP3A4, CYP3A5 and CYP3A7. These efforts have resulted in several publications, revealing the impact of these polymorphisms on antidepressant use, anticoagulation outcomes, diabetes and effectively of breast cancer therapy. Our laboratory also made clear the contribution of pharmacogenetics for the dose prescription of tricyclic antidepressants, showing a clear relation between plasma concentrations, steady state dosage and genetic polymorphisms in CYP2D6. Together with the Dept. Children’s Surgery, the genetic background on different effects of pain medication (morphine, tramadol, paracetamol, propofol) in relation to genetic polymorphisms were studied, again revealing important and potentially clinically relevant associations, which are followed up by an international research program, funded by the European 7th Framework program. The potential role of pharmacogenetics in Oncology was investigated for irinotecan, tamoxifen, paclitaxel and docetaxel. For irinotecan, the impact of UGT1A1 polymorphisms was confirmed, whereas for docetaxel we revealed that there is not a clear role for pharmacogenetics that could be identified at this moment. For tamoxifen, we have provided, together with Dept Internal Medicine and Epidemiology & Biostatistics, important information on the clinical role of CYP2D6 polymorphisms with respect to tamoxifen therapy, on one hand confirming that CYP2D6 poor metabolizers do have a worse prognosis on tamoxifen therapy (for which the FDA is currently adjusting the drug label of tamoxifen), but also illustrating that this relationship is not always present, indicating more reserach is needed to illucidate the background of these discrepancies. In addition, a new, unexpected genetic polymorphisms were identified as a significant predictor of tamoxifen response in breast cancer patients treated with tamoxifen for advanced disease. Because drug metabolizing enzymes also have endogenous substrates, the role of genetic polymorphisms in these genes was investigated in relation to cancer susceptibility in three projects. For prostate cancer, we did find some evidence for of involvement of CYP3A5 in tumor aggressiveness. In addition, the CYP2C19*17 allele proved a prognostic factor for breast cancer.

The department has been active for many years in the ERSPC, biobanking over 400,000 serum samples of prostate cancer patients. An active involvement in development of PSA assays and related tumormarkers, as well as performing PSA measurements for the ERSPC screening program (collaboration with Dept Urology) resulted in the important conclusion that screening on PSA for prostate cancer can significantly decrease prostate cancer mortality with 20%. These results were published in the New England Journal of Medicine in 2009.

Related to the pharmacogenetics of methotrexate, methyl group metabolism is an important aspect of the reserach at our department(project leaders Prof. Dr. Jan Lindemans and Dr. Robert de Jonge). Methyl group metabolism takes place on a roundabout with many entrances and exit routes. The most important vehicles are methionine and homocysteine as substrates, the vitamins folate, B2, B6 and B12 and a collection of enzymes, synthesized from genes many of which demonstrate polymorphisms with effects on activity and stability of their respective enzyme products. Further away from the roundabout there is a relation with nucleotide synthesis, amino acid metabolism, methylation reactions, polyamine synthesis and still other pathways. It is therefore not surprising that the effects of the polymorphisms are found in a variety of disease conditions such as birth defects, atherosclerosis, neurological disease and anemia. It was known that severe homocysteinemia causes bone defects and it has been suggested that mild homocysteinemia might cause age-related osteoporotic fractures. It was finally the outcome of two elaborate studies on subjects from the Rotterdam study (ERGO) and the Longitudinal aging Study Amsterdam (LASA), that demonstrated an overall multivariable-adjusted relative risk of fracture of 1.4 (95% confidence interval 1.2 – 1.6) for an increase of 1SD in the natural log-transformed homocysteine concentration in blood plasma. In a later study we demonstrated that this increased fracture risk was not so much related to the homocysteine raising polymorphism (C677T) in the methyltetrahydrofolate reductase enzyme as such but especially in combination of this polymorphism and low dietary intake of vitamin B2 (riboflavin). We also hypothesized that if the intermediary metabolism of folate is strongly influenced by polymorphisms in the underlying genes of the enzymes involved, this also might be the case for the efficacy and toxicity of folate anti-metabolites such as methotrexate. This indeed proved to be the case in a study on side effects of high-dose methotrexate in children with acute lymphoblastic anemia. In this the MTHFR A1298C polymorphism, the SHMT C1420T polymorphism and the MTRR A66G polymorphism showed a correlation with MTX_induced toxicity. This line of research has been extended with studies on patients with rheumatoid arthritis treated on low dose methotrexate.

Besides being a building stone in protein synthesis, methionine has a vital role in providing methyl groups for protein, lipid and nucleotide methylation. Impairment of methylation capacity with altered DNA-methylation patterns has been thought to be associated with disturbed DNA-regulation and, hence, with malignant disease. Evidence for that hypothesis has always been week. In cooperation with the department of pediatrics we demonstrated, for the first time, an association of the 80G>A polymorphism in the Reduced Folate Carrier – 1 (RFC-1) gene and NNMT IVS -151C>T variants to an increased Acute lymphoblastic Leukemia susceptibility. Most recent research focuses on a further refinement of our observations on methotrexate toxicity in patients with rheumatoid disease. The encountered difficulties as a result of toxic side effects inhibit to exploit the favorable therapeutic effects on the disease activity to the full and we hope, with financial support of the Reuma-foundation, to the specific conditions that predict MTX toxicity with the object of modulating the treatment schedule on an individual basis, guided by genetic information. Herein this project finds its connection to pharmacogenetics with is in a broader sense the common denominator of the research program of the dept. of clinical chemistry.

From a laboratory point of view, the development of validated analyses of genetic polymorphisms in many drug metabolizing enzymes and drug transporters has enhanced the role of the laboratory in pharmacogenetic research. The International federation of Clinical Chemistry nowadays recognizes our laboratory as an International Reference Laboratory for Pharmacogenetics. In this role, the department has a leading role both national and international in the development of pharmacogenetic guidelines, translating pharmacogenetic research into actual patient care. The recognition of the expertise is also apparent from the role of our laboratory in the development and maintenance of a national accessible software system, enabling all pharmacist in the Netherlands to have access to uniform translations of genotypes into phenotypes, including recommendations for adjusting dosages based upon genotype information.

Future plans: special goals and approach

The research on methotrexate, pemetrexed, tamoxifen, taxanes, opioids, anticoagulants and immunosuppressants will be expanded, including a broader candidate gene approach on one hand, and a Genome Wide Analyses approach on the other hand, where possible. Improvement of outcome parameters will be pursued, including development of detection of drug metabolites (Collaboration with Dept. Hospital Pharmacy, Dept. Medical Oncology, dept. neurology and dept. pulmonology) and population pharmacokinetic modelling (collaboration with Dept. Hospital Pharmacy). Prospective intervention studies on use of pharmacogenetics for anticoagulation and taxanes are anticipated. Research will be directed more on pathway pharmacogenetics, in which not only drug transporters and drug metabolizing enzymes will be investigated for a particular drug, but also genetic polymorphisms in relevant receptors and signalling molecules. For research on drug transporters, an LC/MS/MS approach on the cellular level will be developed (collaboration with Dr Luider). Furthermore, efforts will be deployed to translating clinical relevant research findings on pharmacogenetics into robust, high quality pharmacogenetic tests, including recommendations on specific guidelines, quality controls and interpretations. In addition, training programs will be developed as part of the international recognition as Expert Center for Pharmacogenetics.

Most recent publications
  1. van Meurs JB, Dhonukshe-Rutten RA, Pluijm SM, van der Klift M, de Jonge R, Lindemans J, de Groot LC, Hofman A, Witteman JC, van Leeuwen JP, Breteler MM, Lips P, Pols HA, Uitterlinden AG. Homocysteine levels and the risk of osteoporotic fracture. N Engl J Med. 2004 May 13;350(20):2033-41.
  2. Schröder FH, Hugosson J, Roobol MJ, Tammela TL, Ciatto S, Nelen V, Kwiatkowski M, Lujan M, Lilja H, Zappa M, Denis LJ, Recker F, Berenguer A, Määttänen L, Bangma CH, Aus G, Villers A, Rebillard X, van der Kwast T, Blijenberg BG, Moss SM, de Koning HJ, Auvinen A; ERSPC Investigators. Screening and prostate-cancer mortality in a randomized European study. N Engl J Med. 2009 Mar 26;360(13):1320-8.
  3. van der Bol JM, Mathijssen RH, Loos WJ, Friberg LE, van Schaik RH, de Jonge MJ, Planting AS, Verweij J, Sparreboom A, de Jong FA. Cigarette smoking and irinotecan treatment: pharmacokinetic interaction and effects on neutropenia. J Clin Oncol. 2007
  4. de Jonge R, Tissing WJ, Hooijberg JH, Jansen G, Kaspers GJ, Lindemans J, Peters GJ, Pieters R. Polymorphisms in folate-related genes and risk of pediatric acute lymphoblastic leukemia. Blood. 2009 Mar 5;113(10):2284-9.
  5. Schenk PW, van Fessem MA, Verploegh-Van Rij S, Mathot RA, van Gelder T, Vulto AG, van Vliet M, Lindemans J, Bruijn JA, van Schaik RH. Association of graded allele-specific changes in CYP2D6 function with imipramine dose requirement in a large group of depressed patients. Mol Psychiatry. 2008 Jun;13(6):597-605
  6. van Schaik RH, van Agteren M, de Fijter JW, Hartmann A, Schmidt J, Budde K, Kuypers D, Le Meur Y, van der Werf M, Mamelok R, van Gelder T. UGT1A9 -275T>A/-2152C>T polymorphisms correlate with low MPA exposure and acute rejection in MMF/tacrolimus-treated kidney transplant patients. Clin Pharmacol Ther. 2009 Sep;86(3):319-27.
  7. Hesselink DA, van Schaik RH, van der Heiden IP, van der Werf M, Gregoor PJ, Lindemans J, Weimar W, van Gelder T. Genetic polymorphisms of the CYP3A4, CYP3A5, and MDR-1 genes and pharmacokinetics of the calcineurin inhibitors cyclosporine and tacrolimus. Clin Pharmacol Ther. 2003 Sep;74(3):245-54.
  8. Teichert M, van Schaik RH, Hofman A, Uitterlinden AG, de Smet PA, Stricker BH, Visser LE. Genotypes associated with reduced activity of VKORC1 and CYP2C9 and their modification of acenocoumarol anticoagulation during the initial treatment period. Clin Pharmacol Ther. 2009 Apr;85(4):379-86.
  9. van Schaik RHN. CYP450 pharmacogenetics for personalizing cancer therapy. Drug Resist Updat. 2008 Jun;11(3):77-98.
  10. Burger D, van der Heiden I, la Porte C, van der Ende M, Groeneveld P, Richter C, Koopmans P, Kroon F, Sprenger H, Lindemans J, Schenk P, van Schaik RH. Interpatient variability in the pharmacokinetics of the HIV non-nucleoside reverse transcriptase inhibitor efavirenz: the effect of gender, race, and CYP2B6 polymorphism. Br J Clin Pharmacol. 2006 Feb;61(2):148-54.