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Drug Metabolism and Personalized Therapy

Official journal of the European Society of Pharmacogenomics and Personalised Therapy

Editor-in-Chief: Llerena, Adrián

Editorial Board: Benjeddou, Mongi / Chen, Bing / Dahl, Marja-Liisa / Devinsky, Ferdinand / Hirata, Rosario / Hubacek, Jaroslav A. / Ingelman-Sundberg, Magnus / Maitland-van der Zee, Anke-Hilse / Manolopoulos, Vangelis G. / Marc, Janja / Melichar, Bohuslav / Meyer, Urs A. / Nair, Sujit / Nofziger, Charity / Peiro, Ana / Sadee, Wolfgang / Salazar, Luis A. / Simmaco, Maurizio / Turpeinen, Miia / Schaik, Ron / Shin, Jae-Gook / Visvikis-Siest, Sophie / Zanger, Ulrich M.

4 Issues per year


CiteScore 2016: 1.40

SCImago Journal Rank (SJR) 2016: 0.413
Source Normalized Impact per Paper (SNIP) 2016: 0.537

Online
ISSN
2363-8915
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Volume 28, Issue 3

Issues

Congress Abstracts*/Second ESPT Conference “Pharmacogenomics: From Cell to Clinic”

Lisbon, September 26–28, 2013

Published Online: 2013-08-01 | DOI: https://doi.org/10.1515/dmdi-2013-0041

Scientific Committee

Chair: Gérard Siest; Members:

Angela Brand, Roland Bühlmann, Marja-Liisa Dahl, Peter Fitzgerald, Peters Jacobs, Vangelis G. Manolopoulos, Janja Marc, Rui Medeiros, Peter Meier-Abt, Bohuslav Melichar, Urs A. Meyer,

Carolino Monteiro, Helder Mota Filipe, Joao Queiroz, Ron Van Schaik, Maurizio Simmaco, Sophie Visvikis-Siest

Local Organizing Committee

Mafalda Bournon, Pedro Gil, Carolino Monteiro, Sara Torgal, Luis Silva Santos

Pharmacogenomics: From Cell to Clinic

European Society of Pharmacogenomics and Theranostics Second Conference (2013)

Lisbon – Portugal – September 26-27-28, 2013

PROGRAM
Thursday, September 26th Morning

Thursday, September 26th Afternoon

14:30 – 16:30 – FIRST SESSION – Pharmacogenomics and biomarkers in medical oncology: across the spectrum of solid tumors

17:30 – 19:00 – CANCER PHARMACOGENOMICS

Pharmacogenomics: From Cell to Clinic

European Society of Pharmacogenomics and Theranostics Second Conference (2013)

Lisbon – Portugal – September 26-27-28, 2013

Friday, September 27th Morning

09:00 – 11:00 –
SECOND SESSION CLINICAL IMPLEMENTATION OF PHARMACOGENOMICS TESTS

11:50– 13:00 – PHARMACOGENOMICS CLINICAL IMPLEMENTATION

Friday, September 27th Afternoon

14:30 – 17:15 third session transporters and pharmacogenomics

Pharmacogenomics: From Cell to Clinic

European Society of Pharmacogenomics and Theranostics Second Conference (2013)

Lisbon – Portugal – September 26-27-28, 2013

Saturday, September 28th Morning

09:00 – 11:00 – FOURTH SESSION – STEM CELLS AND OTHER NEW TOOLS FOR PHARMACOGENOMICS AND DRUG DISCOVERY

11:30 – 12:30 – SPECIFIC PHARMACOGENOMICS PRESENTATIONS

Pharmacogenomics: From Cell to Clinic

European Society of Pharmacogenomics and Theranostics Second Conference (2013)

Lisbon – Portugal – September 26-27-28, 2013

Saturday, September 28th Afternoon

14:00 – 17:00 – FIFTH SESSION -FROM SYSTEM PHARMACOGENOMICS TO Personalized Medicine

THURSDAY SEPTEMBER 26th MORNING

OPENING LECTURES

Recent progress in molecular methods for pharmacogenomics studies

Daly Ann

The successful implementation of pharmacogenomics requires sensitive methods for the initial detection of pharmacogenomic associations together with simple, rapid and reliable tests for genotyping in the clinic. Many well-established pharmacogenomic associations relating to either drug metabolism or response were detected by studies on single genes already known to have a role in these processes. However, not all interindividual variation in drug metabolism and response or susceptibility to adverse drug reactions is likely to be predictable by studying a single well described candidate gene. Use of genome-wide approaches such as genome-wide association studies (GWAS) and whole exome sequencing allows the detection of multiple genes that contribute to particular phenotypes together with contributions by genes that are not obvious candidates for study. Several examples of the application of GWAS and whole exome sequencing to pharmacogenomics phenotypes will be discussed. Though sequencing and genome-wide genotyping studies have provided interesting new pharmacogenomic data, the methods are complex and may not be suitable currently for routine clinical use. Simple point of care tests to allow genotyping for a small number of polymorphisms rapidly are also valuable and two examples of recently-developed tests relating to coumarin anticoagulants and clopidogrel will be considered.

Pharmacogenomics of endogenous metabolism and its implication for behavior, psychopathology and treatment

Magnus Ingelman-Sundberg, Department of Physiology and Pharmacology, Karolinska Institutet, 171 77 Stockholm, Sweden

Interindividual variation in drug metabolising genes is to a great extent determined by genetic and epigenetic factors. This variability can influence the metabolism of endogenous as well as exogenous substrates. Recent large GWA studies support a link between CYP1A2 polymorphism and blood pressure as well as coffee consumption, and between CYP2A6 polymorphism and cigarette consumption, which in turn appears to influence the lung cancer incidence. Furthermore some studies indicate an association between the number of active CYP2D6 genes and risk for suicide or suicidal behaviour.

We have found an association between depressive mode and the CYP2C19 polymorphism (Sim et al., 2010). In a transgenic model we found that the CYP2C19 gene is expressed in fetal but not in adult brain and causes a reduced hippocampus, altered neuronal composition and a behaviour indicative for anxiety (Persson et al., under re-review). These data will be discussed.

Epigenetic and non-coding (ncRNA) dependent regulation of ADME genes is important and future direction in this novel research field is outlined with respect to our understanding of interindividual differences in drug action (Ivanov et al., 2012). We have in collaboration with the Estonian Genome Center developed a method for analyses of the epigenetics of 174 ADME genes (Ivanov et al., 2013) which identifies the variable epigenetic elements in these genes. In addition we have characterized the 5-hydroxymetylome in human liver in relation to ontogeny and interindividual differences in gene expression (Ivanov et al., submitted). Interestingly we find that in several of the ADME genes up to 25 % of the cytosine modifications are in the form of 5hmC. The 5hmC modifications are primarily in the hepatic active genes and much more abundant in adult as compared to fetal livers. The significance of these modifications for control of ADME gene expression is under review.

References

Sim SC, Ingelman-Sundberg M. Pharmacogenomic biomarkers: new tools in current and future drug therapy. Trends Pharmacol Sci. 2011;32:72 81.

Sim SC, Nordin L, Andersson TM, Virding S, Olsson M, Pedersen NL, Ingelman-Sundberg M. Association between CYP2C19 polymorphism and depressive symptoms. Am J Med Genet B Neuropsychiatr Genet. 2010;153B(6):1160-6.

Ivanov M, Kals M, Kacevska M, Metspalu A, Ingelman-Sundberg M, Milani L. In-solution hybrid capture of bisulfite-converted DNA for targeted bisulfite sequencing of 174 ADME genes. Nucleic Acids Res. 2013;41:e72.

Ivanov M, Kacevska M, Ingelman-Sundberg M. Epigenomics and interindividual differences in drug response. Clin Pharmacol Ther. 2012;92:727-36.

Sim SC, Kacevska M, Ingelman-Sundberg M. Pharmacogenomics of drug-metabolizing enzymes: a recent update on clinical implications and endogenous effects. Pharmacogenomics J. 2013;13:1-11.

LIFE TECHNOLOGIES LUNCH SYMPOSIUM

Your new research companion for Pharmacogenomics

Chair: Peter Jacobs (Gent, Belgium)

Speaker: Dominique Dewolf (Senior Field Application Specialist)

Pharmacogenetics (PGx) refers to the study of genetic differences in drug metabolic pathways and their impact on an individual’s response to specific drugs; both in terms of therapy selection and dose. Although single gene/ drug interactions had been described as early as the 1950’s, recent advancements in genome sequencing technology and use have accelerated our understanding of the impact of germline mutations that lie within pharmacogenetically relevant genes; this work in turn has resulted in an accelerated clinical use of PGx markers to support medical practitioners in making evidence based therapy selection and administration decisions.

Life Technologies has developed an Industry leading technology to support low sample cost, high sample throughput Pharmacogenetic testing. This platform also allows for the flexible addition of assays to new genetic markers, without disrupting existing validated genetic content. To date, this platform has seen rapid adoption and is transforming the Pharmacogenetics testing landscape by making PGx test development economically viable, scalable (from a few to 100’s to 1000’s of samples per day) and adaptable as new clinically relevant genetic markers are translated.

QIAGEN LUNCH SYMPOSIUM

From sample to insight: bringing NGS into clinical routine

Chair: Helene Peyro-Saint-Paul (Marseille, France)

Speaker: Fabienne Hermitte (Marseille, France)

Next-generation sequencing is extensively used in experimental science and has led to significant progress in understanding basic disease mechanisms. However, its adoption in clinical research and diagnostics is still limited by several major challenges:

  • High quality and automated sample preparation process

  • Workflow cost-efficiency, automation and flexibility, related to the increased number of samples to process and need to have a short turn-around time for final result

  • Speed and quality of data analysis and interpretation to obtain a clinically actionable result

QIAGEN is developing a complete automated ecosystem combining 3 components:

  • A pre-analytical portfolio, covering sample isolation and DNA library preparation, and clonal amplification to sequencing and data analysis. The target enrichment step utilizes an easy-to-use and fast (2 hours) multiplex PCR approach with minimal DNA input requirements as low as 20 ng

  • A sequencing workflow, centered around the QIAGEN GeneReader. Based on sequencing-by-synthesis chemistry, the GeneReader technology has the ability to process up to 20 flow cells in parallel through an innovative turntable design flow cell at a time. This unique, random access mode allows molecular biologists to add additional flow cells — even during an ongoing run — significantly speeding up the sample-to-insight time. The absence of need for bar-coding increases the cost-effectiveness of the sequencing phase

  • A data analysis software suite that will allow a fast and accurate interpretation of the NGS data. This will be centered around the Ingenuity suite of web-based applications, including Variant Analysis™ and VCS. These tools are powered by a unique expert-curated Knowledge Base and will allow a rapid identification and prioritization of genetic variants identified through the GeneReader workflow. Both translational researchers and molecular biologists and clinicians will therefore have a rapid access to the biological content behind the NGS data

This complete ecosystem of NGS-dedicated resources, currently in the late development phase, will allow for exome sequencing, whole genome bacterial/virus sequencing, RNASeq, and small RNASeq. It will provide the level of effectiveness, robustness, scalability and data interpretation required for use of NGS in clinical research and diagnostics.

A new IDH1/2 PCR assay for one-step detection of 12 IDH1 and IDH2 mutations in glioma

Speaker: Helene Peyro-Saint-Paul (Marseille, France)

Presence of an isocitrate dehydrogenase (IDH) mutation has a strong diagnostic and prognostic value in glioma, and testing for these mutations will probably be introduced in the next update of the WHO classification system. To date twelve mutations (7 in IDH1 and 5 in IDH2) have been documented in large sample cohorts (ref), and 2 additional mutations have been reported recently as single cases. Current screening for IDH mutations is performed with an IHC assay specific for IDH1 R132H, the most frequent mutation; sequencing is recommended as a second-step test for IHC-negative or –equivocal cases. However, sequencing is not readily accessible in all centres, and its use generally leads to additional delay in providing a comprehensive IDH1/2 mutational status assessment. There is also indication from experimental work that new anti-IDH targeted molecules may be mutation-specific (ref).

In order to address the multiple challenges faced by molecular laboratories and pathologists with IDH1/2 analysis, we designed a qPCR assay able to detect IDH1R132H and 11 rare IDH1/2 mutations in FFPE samples in one single step. PCR clamping was used to detect IDH1 R132H and 11 additional mutations (5 IDH1 R132, 1 IDH1 R100, 5 IDH2 R172). An ARMS design was combined to selectively identify IDH1 R132H, IDH1 R132C and IDH2 R172K.

A cohort of FFPE glioma samples (n=170) was retrospectively analyzed to validate the clinical performance. Samples meeting the assay requirements (age < 10yrs, surface ≥ 50mm2, ≥ 40% tumor cells) were used to compare PCR IDH status to IHC (mIDH1 R132H) status and bidirectional sequencing. Performance on the 11 minor mutations was established using synthetic samples (30% and 45% mut DNA in WT DNA).

Assay sensitivity varied across mutations with LOD <5% for 11/12 mutations (mean=3.3%). From the first 120 clinical samples analyzed, 103 were <10 yrs (glioblastomas: 48/103). IDH status was successfully obtained by PCR for these 103 samples. All IHC positive cases (n=45) were concordantly identified as R132H by PCR. One R132H case detected by PCR was negative by IHC (negative concordance 98%). PCR additionally detected 9 rare mutations (8 IDH1, 1 IDH2). Moreover, the kit produced 100% correct results on the synthetic samples with rare mutations.

This new one-step IDH1/2 PCR assay showed a 100% technical success rate and is more sensitive than published references for bidirectional sequencing. Positive concordance with IHC detection for IDH1 R132H was 100%. Complete validation results including sequencing data will further document the value of this new assay to screen in one step for 12 IDH12 mutations.

THURSDAY SEPTEMBER 26th AFTERNOON

FIRST SESSION – Pharmacogenomics and biomarkers in medical oncology: across the spectrum of solid tumors

Integrating Pharmacogenetics testing at the bedside: Yes we can!

Joseph Ciccolini - Pharmacokinetics Unit - Inserm U911 Aix-Marseille Univ & University Hospital of Marseille - Marseille, France

Developing personalized medicine is an irreversible trend in clinical oncology. A variety of tests are now made available to better predict treatment efficacy, through the screening of genetic or molecular dysregulations affecting tumor cells, and possibly leading next to poor clinical outcome in patients with cancer. Search for genetic or molecular determinants of response prior to starting treating patients, especially with costly targeted therapies or biologics, is now a widely spread clinical practice. Beside pharmacogenomics testing at the tumor level, constitutive genetic deregulations possibly affecting the pharmacokinetics of anti-cancer agents is a rising concern, which has not made its way yet from experimental pilot studies to routine clinical practice. Genetic polymorphisms affecting liver enzymes responsible for drug disposition, or affecting membrane transporters implicated into drug distribution, are as critically important as mutations at the tumor level for achieving clinical benefit eventually. It is widely acknowledged today that genetic polymorphisms affecting DPYD, UGT1A1, TPMT or CDA are associated with life-threatening and sometimes lethal toxicities with 5-FU/capecitabine, irinotecan, 6-mercaptopurine or gemcitabine, respectively. However, no official recommendations for testing patients prior to administering these cytotoxics has been issued by health authorities and regulatory agencies, despite countless reports demonstrating that the efficacy/toxicity balance of these drugs is dramatically worsen in mutated patients. Despite the lack for official recommendations, when preliminary testing is carried out and fully integrated as part of routine clinical practice in oncology, immediate benefit is achieved since the incidence of severe or lethal toxicities is sharply reduced through the use of adaptive dosing strategies. Integrating pharmacogenetics covariates into dosing algorithms should be the final step that remains to be taken to achieve personalized medicine in oncology.

The Pharmacogenomic profile and the management of castration-resistant prostate cancer: Are we ready for the new drugs personalized therapy?

Rui Medeiros, IPO, Porto, Portugal. ruimedei@ipoporto.min-saude.pt

Pharmacogenomics, Transcriptomics, Genomics, Metabolomic, Interactomics, Immunogenomics, Genomics, Proteomics an other omics are currently more common nouns in the field of Biology and Medicine.

We consider that "..omics" may be the comprehensive analysis of biological systems whether you are focusing your attention in DNA, RNA, Proteins, Metabolites or other molecules. Cancer development and other aging related diseases are under the dependence of high complexity systems that may also influence their effective response to therapeutics.

Since we made the introduction of a Pharmacogenomics and Molecular Epidemiology Class in the Faculty of Medicine of Porto (2004) and in the Biomedical Sciences Institute (ICBAS, 2006) an increasing number of research reports have been published demonstrated the importance of this "omics" research field in Medicine, and particularly in Oncology. A comprehensive review will be presented pointing some research lines.

Castration resistance is a life-threatening event that may develop in prostate cancer patients with advanced disease following hormonal castration therapy (HCT). Androgen deprivation therapy (ADT) through surgical or chemical castration has been the standard of care for the initial treatment of metastatic prostatic adenocarcinoma. Most patients with metastatic prostate cancer experience disease progression despite castration. Castrate-resistant prostate cancer (CRPC) is principally responsible for prostate cancer mortality, and fewer than 20% of patients with CRPC survive beyond 3 years. Current understanding of the molecular mechanisms behind resistance to hormonal castration suggests a role for androgen receptor signaling and bioavailability of androgens. However, alternative pathways may also be considered. Published results suggest that genetic polymorphism may influence response to treatment and time to resistance to hormonal castration therapy. The strategy for using these genetic variants in the definition of a pharmacogenomic profile of prostate cancer resistant to hormonal castration is under discussion.

Pharmacogenomics and Biomarkers in medical oncology: Lung Cancer

Guy Berchem - Medical Oncologist & Head Experimental Hemato-Oncology Laboratory CRP Santé – Luxembourg Hospital - Luxembourg

Lung cancer is the first cause of cancer related death in men and in some countries even in women meanwhile. It is the most common and deadly malignant tumor worldwide. The main challenge in this disease is to develop strategies in order to improve the poor survival rate (5-year survival of approximately 15%) and to develop novel strategies to better stratify high-risk populations for early diagnosis as well as to select the adequate treatment for different lung cancer subsets.During the last years, a lot of progress has been made in the identification of molecular aberrations of lung cancer. These molecular abnormalities can be used as drug targets, as predictive biomarkers for selection of targeted therapies as well as for disease prognosis and prediction of classical chemotherapy regimens. These recent advances in NSCLC targeted therapy require the analysis of a panel of molecular abnormalities in tumor specimens, including gene mutations (e.g., EGFR, KRAS, BRAF, PI3kinase…), gene amplification (e.g., MET, FGFR1), and fusions (e.g., EML4-ALK, ROS, RET), as well as DNA repair enzymes (e.g., ERCC1, RRM1, AEG1…).In addition to the gene based strategies, lung cancer has even been added to the list of immunotherapy sensitive tumors lately, mainly due to the emergence of checkpoint inhibitors like anti-CTLA4, PD1 and PDL1 antibodies.In 2011 we initiated a personalized Lung cancer program aiming at bringing the advantages of molecularly targeted lung cancer treatment to the Luxembourg patient now. Within this program we have organized the collection of diagnostic FFP blocs, the molecular analysis as well as the collection of patient data. The annotated molecular results with treatment suggestions are then returned to the treating physician and the patient outcome correlated with the molecular data and the treatment. Preliminary data on molecular alterations and clinical correlations will be presented.

Pharmacogenomics in colorrectal cancer

David Páez López-Bravo - Hospital de la Santa Creu I Sant Pau - C/ Mas Casanovas 90 - 08041 Barcelona, Spain

Chemotherapeutic and biological agents used in colorectal cancer present a disparity in terms of efficacy and are frequently associated with severe adverse reactions that compromise the efficacy of treatment. Predicting this efficacy/toxicity could enable therapy to be tailored. Genetic variations have been associated with efficacy/toxicity in patients treated with fluoropyrimidines, oxaliplatin, irinotecan and bevacizumab and cetuximab. Complexity of treatment and variability in toxicity classifications make it difficult to compare studies. This presentation analyzes the association between different pharmacogenomic biomarkers and the efficacy/toxicity of the treatme used in colorrectal cancer. In addition, the state-of-the-art and future perspectives are discussed.

Association of glutathione S-transferase gene variations, busulfan exposure and clinical outcomes in children receiving intravenous busulfan before hematopoietic stem cell transplantation.

Ansari Marc, Uppugunduri Chakradhara Rao S, Rezgui Mohamed Aziz, Théoret Yves, Mezziani Samira, Vachon Marie-France, Desjean Catherine, Rousseau Julie, Labuda M, Przybyla C, Duval Michel, Champagne Martin A, Peters Christina, Bittencourt Henrique, Krajinovic Maja

Busulfan (BU) is a key compound of conditioning regimens in children undergoing hematopoietic SCT (HSCT). Inter-individual differences in BU pharmacokinetics (PKs) might affect BU efficacy and toxicity. Intravenous BU combined with therapeutic drug monitoring-guided dosing is associated with better event-free survival (EFS) and lower transplant-related mortality (TRM). But optimal target steady state concentration (Css) of Bu in children undergoing HSCT and predictable genetic markers of Css remains unclear. This study includes 75 children receiving intravenous (iv) BU in 16 doses with first dose assigned based on age. BU first dose pharmacokinetic parameters were estimated from plasma levels of BU measured at six time points by high-performance liquid chromatography method. Further, doses were adjusted at the 5th dose level to a target Css of 600-900 ng/mL. Clinical outcomes were recorded during follow up of 5 years. Institute Review Board approved the study, and informed consent was obtained from all patients or parents prior to their enrollment. As BU is mainly metabolized by glutathione S-transferase (GST), we investigated the relationship between GSTA1, GSTM1 and GSTP1 genotypes with first-dose BU PKs, and the relationship with HSCT outcomes in a subset of 69 children receiving myeloablative conditioning regimen. Cumulative incidence of overall survival (OS), EFS, TRM, acute graft-versus host disease (aGVHD), veno-occlusive disease (VOD) and other toxicities in relation to Css of BU, GST genotypes were analyzed using Kaplan-Meier curves in univariate and Cox proportional hazards regression model in multivariate analysis. After first dose, median Css was 578 ng/mL (325-1227). Forty-one patients had > 10% BU iv dose increased. First-dose BU Css > 600 ng/mL was associated with a higher non relapse mortality (NRM, p<0.001) and grade 2-4 aGVHD (p=0.04), a lower EFS (p<0.001) and OS (p=0.001). We observed the association of GSTM1 null genotype with higher BU exposure and lower clearance in patients older than 4 years (P 0.04). In accordance with the suggested functional role, GSTA1*A2 haplotype was associated with lower drug levels and higher drug clearance (P 0.03). Gene-dosage effect was also observed (P 0.007). GSTA1 haplotypes were associated with HSCT outcomes. Patients with two copies of haplotype *A2 had better EFS (P=0.03). In contrast, homozygous individuals for haplotypes *B and *B1 had higher occurrence of VOD (P=0.009). GSTM1 null individuals older than 4 years had more frequent occurrence of aGVHD (P=0.03). In conclusion, we demonstrated a significant correlation between the first-dose pharmacokinetic of BU, NRM, and survival, probably due to toxicity. We also have shown that GST gene variants influence first dose BU PK and outcomes of HSCT in children. A model for the dosage adjustment with the inclusion of genetic and non-genetic factors initiating from first dose onwards should be evaluated in a future prospective validation cohort.

Anticancer drug-relevant solute carrier transporters and their role in resectable pancreatic cancer

Mohelnikova-Duchonova Beatrice, Soucek Pavel, Brynychova Veronika, Oliverius Martin, Hlavsa Jan, Honsova Eva, Mazanec Jan, Kala Zdenek, Melichar Bohuslav

Objectives: The aim of this study was to investigate the prognostic potential of major solute carrier transporters (SLCs) known for their ability to transport nucleoside analogs and platinum drugs on a well-defined group of pancreatic cancer patients in the context of clinical-pathological characteristics and the KRAS mutation status of tumors.

Methods: Tumors and non-neoplastic pancreatic tissues were obtained from 32 histologically verified patients with pancreatic ductal adenocarcinoma. The transcript profile of SLCs was assessed using quantitative real-time PCR. KRAS mutations in exon 2 were assessed by high resolution melting analysis and confirmed by sequencing.

Results: SLC22A3 was upregulated and SLC22A1, SLC22A2, SLC22A11, SLC28A1, SLC28A3 and SLC29A1 were downregulated when compared with non-neoplastic pancreatic tissues. Moreover, significantly lower levels of SLC22A1, SLC22A11 and SLC29A1 were found in tumors with angioinvasion. There was also a significantly higher transcript level of SLC28A1 in tumors with regional lymph nodes affected by metastasis. The study found that a high expression of SLC22A1 or SLC28A1was significantly associated with poor overall survival in unselected patients. In contrast, a high expression of SLC22A3 or SLC29A3 was significantly associated with longer overall survival in patients treated with nucleoside analogs. Finally, SLC levels were not found to be associated with KRAS mutation status in exon 2.

Conclusions: The results of this study revealed a considerable variability in the expression of SLC transporters between tumor and normal human pancreas tissues which may modify the outcomes of patients treated with nucleoside analogs- and platinum-containing regimens. The expression of some SLCs predicted the clinical outcomes of resectable pancreatic cancer.

Supported by the Czech Science Foundation grant P301/12/1734

CANCER PHARMACOGENOMICS

Prognostic and predictive biomarkers in colorectal cancer based on somatic mutation obtained by deep sequencing

Andre Rosenthal - Signature Diagnostics AG, Hermannswerder 20A, 14473 Potsdam, Germany

Abstract missing

Pharmacogenetic determinants of glucocorticoid sensitivity in childhood acute lymphoblastic leukemia

Dolzan Vita, Prezelj Neza, Mercun Nusa, Faganel Kotnik Barbara, Debeljak Marusa, Jazbec Janez

Background: The response to glucocorticoids during the induction of remission is an important prognostic factor in childhood acute lymphoblastic leukemia (ALL). It is commonly accepted that genetic variability of drug metabolizing enzymes, transporters and drug targets may influence treatment response, but only a limited number of studies investigated genetic determinants of glucocorticoid sensitivity in ALL. Beside genetic variability of glucocorticoid receptor NR3C1 (GR) that mediates the intracellular action of glucocorticoids, genetic variability of P-glycoprotein (ABCB1) and glutathione S-transferases (GST) may influence their bioavailability and thus influence treatment response. Our aim was to investigate genetic factors that could influence the outcome of the induction treatment of childhood ALL with glucocorticoids.

Methods: In total 86 Slovenian children and adolescents with ALL treated with glucocorticoids during the induction phase were genotyped for common polymorphisms in NR3C1, ABCB1, GSTM1, GSTT1 and GSTP1 genes. Logistic regression was used to assess the influence of polymorphisms on the resistance (> 1000 blasts/μL on day 8) or poor response (> 100 blasts/μL on day 8) to glucocorticoid treatment, and on the risk of relapse, event (relapse or death) and exitus. Cox regression was used to determine their influence on event-free survival (EFS).

Results: On day 8 of prednisone treatment 6 patients (7,0 %) had > 1000 blasts/μL, while 26 (30,2 %) patients had > 100 blasts/μL The protective effect of NR3C1 rs33388 A allele against resistance to induction treatment was not statistically significant (OR 0,210; 95 % CI 0,036-1,236, p = 0,084). The carriers of GSTP1 rs1695 G allele had increased risk of poor response to prednisone treatment (OR 7,040; 95 % CI 1,826-26,618, p = 0,004), increased risk of event (relapse or death) (OR 3,75; 95 % CI 0,986-14,268; p = 0,053) and shorter EFS (HR 3,382; 95 % CI 1,028-4,172; p = 0,042). These associations remained significant in haplotype analysis. GSTP1 GC haplotype, present in 33 % of our ALL patients, increased the risk of poor response to prednisone (OR 3,963; 95 % CI 1.252-12.544; p = 0,019), the risk of event (OR 3,678; 95 % CI 1.308-10.341; p = 0,014) and exitus (OR 4,810; 95 % CI 1,157-19,988; p = 0,031). ABCB1 GT haplotypalso increased the risk of poor response to prednisone (OR 5,055; 95 % CI 1.138-22.465; p = 0,033).

Conclusions: Our results suggest an important role of GSTP1 rs1695 polymorphism in glucocorticoid sensitivity and treatment outcome in childhood ALL.

Genetic risk factors of anthracycline-induced cardiotoxicity – relevant polymorphisms identified in enzymes and transporters of anthracycline pharmacokinetics

Kutszegi Nora, F. Semsei Agnes, Lautner-Csorba Orsolya, Hegyi Marta, Szalai Csaba, Kovacs Gabor T. Erdelyi Daniel J.

Aims: The main dose limiting side-effect of anthracyclines is late cardiotoxicity. Survivors of anticancer therapy have increased risk for cardiovascular problems and have higher such mortality. Subclinical changes may become crucial in case of later accompanying diseases affecting the cardiovascular system, or these changes can precede severe late onset cardiac failure. Identifying patients with altered tolerance to anthracyclines would provide great clinical benefit.

Methods: We studied 164 paediatric acute lymphoblastic leukaemia (ALL) patients who had been treated with ALL BFM protocols. They had cardiac ultrasound scans with a mean follow up of 6.4 years after anthracycline therapy. Left ventricular function was assessed as fractional shortening (LVSF). Germline genotypes of 19 single nucleotide polymorphisms (SNPs) in the ABCC1, CBR1, CBR3 and AKR1A1 genes were measured. Multifactorial general linear model was used to test for associations.

Results: Patients with ABCC1 rs246221CT/TT genotype had lower LVFS at the time of the latest echocardiography compared to CC patients (38.4% and 40.7% respectively, p=0.027). Those with AKR1A1 rs2088102CC genotype had lower LVFS than those harbouring at least one T allele (36.9% and 39.1% respectively, p=0.013). Further SNPs showed no association with left ventricular function.

Conclusion: Our results suggest that the ABCC1 rs246221 and the AKR1A1 rs2088102 variations are associated with altered left ventricular function in late survivors of childhood acute lymphoblastic leukaemia. The identified early subclinical changes.

FRIDAY SEPTEMBER 27th MORNING

SECOND SESSION – CLINICAL IMPLEMENTATION OF PHARMACOGENOMICS TESTS

Introduction

Ron H.N van Schaik - Department of Clinical Chemistry (AKC), Room Na-415, Erasmus MC, Gravendijkwal 230, 3015CE Rotterdam, the Netherlands

Two decades of clinical pharmacogenetic testing - where do we stand?

Marja-Liisa Dahl – Department of Laboratory Medicine, Karolinska Institutet – Stockholm, Sweden.

Drug response is a multifactorial trait, influenced by genetic, environmental, and disease determinants that affect both drug kinetics (PK; absorption, distribution, metabolism, excretion) and drug effects at the target site or beyond (pharmacodynamics, PD). Great expectations have during the last decades been placed on identification of genetic factors influencing this variability. Much of the early pharmacogenetic research was focused on PK, especially metabolic variability, with the potential to explain and predict drug plasma concentration profiles over time, and consequently, individualized dose prediction. Consequently, a number of pharmacogenetic tests for drug metabolic capacity (e.g. CYP2D6, 2C19, 2C9, UGT1A1, TPMT) were introduced and are now increasingly available for clinical use. More recently, tests to identify individuals at risk of adverse effects (e.g. HLA-B*5701 and abacavir hypersensitivity) and tests predictive of drug response in e.g. oncology have been introduced. Pharmacogenetic testing as a health care service will be discussed based on twenty years of experience at Karolinska University Hospital.

Improving cancer drug therapy through the patient genomes

Federico Innocenti, MD, PhD - University of North Carolina at Chapel Hill - Institute of Pharmacogenomics and Individualized Therapy innocent@unc.edu

Genomic explorations into the heritable and acquired components of the human and cancer genomes have elucidated the genetic basis of the interindividual differences in drug response in cancer patients. With few notable examples, the road to application of genomics to the clinic at the bed side remains tortuous. At the University of North Carolina (Chapel Hill, NC, USA) an infrastructure called UNCseq has been established for providing results to cancer patients based upon sequencing of their cancer genomes (both acquired and germline). This presentation will illustrate the most prominent advances in the sequencing of the cancer genome so far. It will also describe the infrastrcure available at our institution for identifying actionable and druggable variants for intervention and for reporting results to patients and physiciansmay contribute to a polygenic disorder that evolves over a longer time and manifests in congestive heart disease later.

Prospective clinical trials on coumarin anticoagulant pharmacogenomics: the moment of truth

Vangelis G. Manolopoulos Laboratory of Pharmacology, Democritus University of Thrace Medical School, Alexandroupolis, Greece, and Clinical Pharmacology Unit, Academic General Hospital of Alexandroupolis, Greece

email: emanolop@med.duth.gr

Coumarin derivatives such as warfarin, acenocoumarol and phenprocoumon constitute the world-wide oral anticoagulant treatment of thromboembolic disorders. Response to therapy to these drugs exhibits significant variation among patients. A great proportion of this variation is due to genetic background of individuals. Specifically, variations both in the genes of cytochrome P450 enzyme CYP2C9 and vitamin K reductase VKORC1 influence individual responses to anticoagulant therapy. CYP2C9*2 and *3 variant alleles result in decreased CYP2C9 enzymatic activity affecting coumarin pharmacokinetics, while VKORC1 -1639G>A polymorphism influence pharmacodynamics response to coumarins. It appears that lower doses of coumarins may be best for patients with variations in one or both of these genes and efforts are made to incorporate this knowledge in currently used dosing regiments. Towards this direction, pharmacogenetic-based dosing algorithms are currently tested in large prospective, randomized, pharmacogenetic clinical trials both in Europe and the USA, including EU-PACT (European Union-Pharmacogenetics of Anticoagulant Therapy) Trial. Some of these trials have been completed and their results have been announced or will soon be announced. It is expected that the results of these trials will provide a solid basis for broadly implementing genotype-guided dosing of anticoagulant therapy in clinical routine practice. This should lead to reduced bleeding or thrombotic events and increased effectiveness of anticoagulant agents.

Diagnostics for Alzheimer’s disease (AD): from research to practice

Edith Schallmeiner – Rotkreuz, Switzerland - Roche Diagnostics

The development of medications to prevent or delay AD progression has so far met with limited success, perhaps because treatment needs to be initiated early in the disease continuum in order to provide the greatest long-term benefits. There is therefore a significant need for diagnostic tools that can accurately identify patients early in the disease process (including those at high risk of developing AD) for inclusion in clinical trials, and for subsequent use in clinical practice.

Low cerebrospinal fluid (CSF) concentrations of Aβ1–42 peptide (Aβ42), in combination with high CSF concentrations of total tau (T-tau) and phosphorylated tau (P-tau) are biomarkers that are believed to be predictive of cognitive decline in prodromal AD patients. Although imaging techniques are also under evaluation, biomarkers based on CSF provide a immunoassay based approach for the identification of patients at risk of AD development. However, there are concerns regarding the wide inter-laboratory variations in the results seen with the immunoassays currently available and standardization efforts are ongoing. The development of highly reproducible, fully automated assays may enhance the utility of CSF biomarkers for clinical research and more widespread clinical use in AD. Efforts to find reliable biomarkers for AD in peripheral blood have had little success to date.

Although not a causative genetic mutation, apolipoprotein ε4 (APOE ε4) is a well-recognized risk modifier for the development of AD, with the risk increasing further in those who inherit two compared with one copy of the gene. SUBJECTS who meet guideline criteria for mild cognitive impairment and are also APOE ε4 positive are more likely to progress to AD than those who do not carry the gene.

This is an exciting time for the development of diagnostic tests in AD and it is hoped that their application in clinical trials will help to more fully define the potential benefits of disease-modifying treatments for this devastating disease.

Pharmacogenetics and the treatment of infectious diseases in the Developing World – malaria as a translation poof of concept

Gil JP Drug Resistance Unit, Section of Pharmacogenetics, Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden

The chemotherapy of infectious diseases poses specific challenges for the translation of pharmacogenetics knowledge for treatment optimization. The first issue has to do with time: in general, an infectious disease is met in or near its acute phase. In the case of life threatening infections treatment has to be swiftly provided, as it represents a race against the fast expansion of the pathogen. Secondly, the pharmacogenetics of infectious diseases involves not only the human host, but also the contributions of the invading organism (s). Information from the former will be applied for a preview of the individual disposition of the drug, while the later will influence the pharmacodynamics of the treatment.

Such challenge need new technologies; specifically point of care, affordable devises that allow a fast output of information in order to support decisions on therapeutic options.

Such a technology is presently being developed by a private sector-academia consortium, under the umbrella of the European Commission Framework 7. The network involves the nanotechnology venture QuantumDX, The University of London, The University of Tubingen and the Karolinska Institute. The aim is to produce a low cost hand held device allowing point of care genetic analysis. The management of deadly drug resistance Plasmodium falciparum malaria represents the elected proof of concept. The fact that the analysis has to be compatible with both, the necessary low cost of malaria therapy (< 2 €), and the demand of being applicable to low income settings sets further challenges.

Although aimed in this first phase for malaria and the Developing World, it is expected for the lessons learnt in this project to provide a smooth back translation for the further development of this technology concerning Industrialized world health care settings and demands.

Iris Grossman (Netanya, Israel) – Teva Pharmaceutical Industries

Abstract missing

PHARMACOGENOMICS CLINICAL IMPLEMENTATION

Personalized Pharmacogenetics

Charles R. Cantor, Chief Scientific Officer SEQUENOM, Inc. San Diego, CA, USA 858 202-9013 ccantor@sequenom.com

Genetic variations that predict drug response are among the very few human traits that are relatively simple. In addition to the traditional ADME markers that are easily measured in the germ line, somatic DNA changes in cancer are often extremely informative in predicting drug efficacy. These tumor-specific markers cannot be seen in the germ line, but they are detectable in tissue biopsies and increasingly in plasma where they arise from apoptotic events in the tumor. Several recent examples show that detection of mutations in plasma is an excellent way to monitor cancer therapy, and it is likely that such analyses will see routine clinical use within a few years. In addition it appears that tumor- specific epigenetic markers can also be detected in plasma and are useful in patient management. It should be worthwhile to search for equivalent epigenetic markers detectable in plasma as a way of monitoring therapeutic efficacy in a range of other diseases.

Synergy between clopidogrel and calcium-channel blockers for blood pressure regulation possibly involves CYP2C19 polymorphism

Stathopoulou Maria, Monteiro Pedro, Shahabi Payman, Penas-Lledo Eva, El Shamieh Said, Silva Santos Luis, Thilly Nathalie, Siest Gerard, Llerena Adrian, Visvikis-Siest Sophie

Background: Interactions between clopidogrel and calcium-channel blockers (CCB) have been identified in regards to antiplatelet activity but not for other cardiovascular variables. The aim of the study is the determination of the effect of joint use of clopidogrel and CCB on blood pressure levels (BP). Furthermore, the effect of the rs4244285 polymorphism of CYP2C19 gene on BP levels was tested.

Methods and Findings: For this cross-sectional observational study, patients consecutively admitted to the intensive coronary care unit of the Coimbra University Hospital (Portugal) between May 2004 and July 2011, for acute myocardial infarction or unstable angina, were recruited. Patients for whom pre-admission medication data was not available were excluded. Final sample consisted of n=2,322. The genetic assessment was performed on a healthy European population (n=2,155). Data collected in both samples included age, gender, weight, height, BP and data for pharmacological therapy prior to admission was recorded, during the procedure of medical history assessment for the cardiovascular patients. Associations were assessed using linear regression models adjusted for relative co-factors. Clopidogrel use was significantly associated with lower diastolic blood pressure (DBP) (P= 0.034, beta= -1.47mmHg) in the total sample and in the CCB group (P= 0.030, beta= -3.07mmHg). The genetic analysis revealed an association between rs4244285 and both systolic and DBP (P=0.008, beta: -1.69mmHg and P=0.003, beta: -1.28mmHg respectively) in the healthy population. Due to study design, causal relationships and implicated mechanisms could not be assessed.

Conclusions: A synergistic effect between clopidogrel and CCB for reduction of BP levels was observed and a novel genetic variant related to BP was identified. This effect and its possible mechanisms should be investigated in future studies.

Formation of a TPMT nomenclature committee - bringing order and consensus around known and novel TPMT sequence variants

Lindqvist Appell Malin, Coulthard Sally

The drug metabolizing enzyme thiopurine methyltransferase (TPMT) has become one of the best examples of pharmacogenomics to be translated into routine clinical practice. TPMT metabolizes the thiopurines 6-mercaptopurine, 6-thioguanine and azathioprine, drugs widely used for treatment of acute leukemias, inflammatory bowel diseases and other disorders of immune regulation. Since the discovery of genetic polymorphisms in the TPMT gene, many sequence variants which cause a decreased enzyme activity have been identified and characterized. Increasingly, to optimize dose, pre-treatment determination of TPMT status before commencing thiopurine therapy is now routine in many countries. Novel TPMT sequence variants are currently numbered sequentially using PubMed as a source of information; however this has caused some problems as exemplified by two instances in which authors’ articles appeared on PubMed at the same time, resulting in the same allele numbers given to different polymorphisms. Hence, there is an urgent need to establish an order and consensus to the numbering of known and novel TPMT sequence variants. To address this problem, a TPMT nomenclature committee was formed in 2010, to define nomenclature and numbering of novel variants for the TPMT gene. A total of 16 scientists, spread around the world, all working within the area of TPMT pharmacogenetics are members of the committee, many of which has themselves discovered novel TPMT alleles. A website www.imh.liu.se/tpmtallelesserves as a platform for the work of the committee. Researchers are encouraged to submit novel TPMT alleles to the committee for designation and reservation of unique allele numbers. The committee has decided to renumber two alleles: nucleotide position 106 (G>A) from TPMT*24 to TPMT*30 and position 611 (T>C, rs79901429) from TPMT*28 to TPMT*31. Nomenclature for all other known alleles remains unchanged.

Relevance of CYP2C19 genotypes in Nevirapine biotransformation

Grilo Nádia M., Marinho Aline T., Naranjo María Eugenia G., Caixas Umbelina, Branco Teresa Antunes Alexandra M. M., Marques M. Matilde, Monteiro Emília C., Llerena Adrián, Pereira Sofia A.

Nevirapine (NVP) is one of the most prescribed anti-HIV drugs, partly due to its low cost (Lockman et al., 2007) and favorable metabolic profile (Clotet et al., 2003). However, NVP use has been associated with severe idiosyncratic hepatotoxicity and cutaneous hypersensitivity (Cattelan et al., 1999). Although the mechanism underlying NVP toxicity is still uncertain, several studies in vitro (Antunes et al., 2010), in animal models (Chen et al., 2008) and more recently in man (Caixas et al., 2012) have suggested that bioactivation of the Phase I NVP metabolite, 12-hydroxy (OH)-NVP, to reactive electrophiles (e.g., 12-sulfoxy-NVP) is involved. Both CYP3A4 and 2B6 have been identified as the major contributors to the biotransformation of NVP into 12-OH-NVP and recent evidence also supports the involvement of CYP2C19 (Lehr et al., 2011). Moreover, this CYP isoenzyme has been linked to NVP-GSH adduct formation (Wen et al., 2009). The purpose of this study was to analyze the influence of CYP2C19 variant alleles on NVP/12-OH-NVP metabolic ratio (MR). NVP biotransformation was studied in a population of 51 HIV-infected patients (63% were men; 65% were Caucasian; 49±1.7 years old). All eligible patients were adults with documented HIV-infection who had received continuous treatment with NVP-based combined antiretroviral therapy (400 mg once daily) for more than 1 month. The protocol of the clinical study received prior approval from the Hospital Ethic Committees and patients gave their written informed consent. Plasma concentrations of NVP and 12-OH-NVP were determined by high-performance liquid chromatography. CYP2C19 genotyping for the variant alleles CYP2C19*2 (poor metabolizer) and *17 (ultra-rapid metabolizer) was carried out by real-time PCR. One subject was homozygous and 4 individuals were heterozygous for CYP2C19*2. In respect to CYP2C19*17, one subject was homozygous and 7 were heterozygous. An uncertain result in the genotyping analysis was found for 27% of the individuals. NVP concentrations were higher (ANOVA, P < 0.05) for CYP2C19*2 (median (IQR) CV: 5.49 (4.98-6.17) mg/L; 13%) than for CYP2C19*1 (wild-type; 4,16 (3.59-4.77) mg/L; 21%) carriers. No differences were found for CYP2C19*17 (4.75 (2.56-5.21); 34%) when compared to wild-type carriers. For the 12-OH-NVP concentration, no differences were found when comparing CYP2C19*2 (0.485 (0.301-0.543) mg/L; 30%) and CYP2C19*17 (0.403 (0.250-0.454) mg/L; 34%) with CYP2C19*1 (0.352 (0.292-0.476) mg/L; 77%). In the case of NVP/12-OH-NVP MR, no differences were found when comparing CYP2C19*2 (10.9 (9.7- 13.9); 22%) and *17 (10.6 (9,6-12.8); 17%) with CYP2C19*1 (11.6 (10-13.5); 44%). These results support the hypothesis that CYP2C19 has a role in NVP biotransformation in humans, although it seems not to be involved in the 12-hydroxylation of NVP.

RANDOX LUNCH SYMPOSIUM

Application of Biochip Array Technology for Multiplex Pharmacogenomic Profiling

Chair: John Lamont – Randox Laboratories Ltd, Crumlin, Northern Ireland

The ‘Rheumastrat’ biochip array; a multiplex SNP array for the stratification of therapy response in Rheumatoid arthritis patients

Cathy M McGeough*, Helena Murray, Mark Latten, David Gibson, Mary Slevin, Gary Wright, Philip Gardiner, Oliver Fitzgerald, Tony Bjourson, Martin A Crockard

*University of Ulster - Northen Ireland

Background: Personalized therapy for rheumatoid arthritis (RA) patients remains an unmet clinical need in the management of this disease. Although, biologic treatments such as anti-Tumour Necrosis Factor (anti-TNF) therapy have shown remarkable efficacy, the life-changing effect of these expensive therapies is restricted to the 60-70% of patients who respond. The immune reaction to anti-inflammatory therapy is thought to be influenced by many genes which cumulatively contribute to a threshold for response. This study examines the polygenic contribution of a panel of genes related to poor outcome and response to therapy using Biochip Array Technology™.

Materials and methods: DNA was extracted from peripheral blood mononuclear cells of RA patients treated with biologic therapy. DNA based probes, primers and multiplex PCR methods were designed and optimised to develop the biochip, which simultaneously assess all targets of interest, within 3 hrs. The Rheumastrat biochip was developed using standard control samples of known genotype and validated by reference methods (PCR-sequence specific probes and sequencing).

Results: On-chip 21-plex analysis is now available and genotyping has been completed for all patient samples. The biochip provides detailed allelic information and creates a genotypic profile for each patient which can be used to determine their likely clinical treatment outcome.

Conclusions: The Rheumastrat biochip aims to assist in the clinical management of RA through patient DNA analysis. This approach promotes timely and efficacious treatment for the patient, to improve their quality of life, and positively impact healthcare budgets.

The importance of VEGF in pharmacogenomics

Sophie Visvikis-Siest - UMR INSERM U1122; IGE-PCV, University of Lorraine, Nancy, France

Vascular endothelial growth factor (VEGF) is implicated in angiogenesis, lymphangiogenesis, vascular permeability, and hematopoiesis. It is associated with numerous pathologies including cardiovascular diseases, diabetes, several types of cancer, cognitive decline and dementia, and reproductive and immune-inflammatory disorders. Also, VEGF inhibitors are used in cancer, macular degeneration and rheumatoid arthritis treatment, showing however significant toxicity. The biological determinants and the influence of the genetic component of VEGF are still not well described.

Consequently, we specifically developed an integrative systems biology strategy for improvement of this biomarker in clinical and pharmacogenomics studies.

The influence of pre-analytical variables on VEFGA circulating protein concentrations and on gene expression was an important information to search and to take into account.The biological determinants of VEGF interindividual variation and the reference values for VEGF plasma levels were determined. A high heritability of this trait, 60%, was estimated in the STANISLAS cohort giving us the needed arguments to continue for a deep characterization of the genetic component of VEGF levels.

Therefore, we searched, by a Genome Wide Association Study (GWAS), the VEGF genetic variants and the interconnexions of these biomarkers with other disease-associated molecules in healthy populations.

The GWAS was performed in 3,527 healthy participants (Framingham Heart Study) and the most significant results (P <5×10-8) were replicated in 1,727 individuals (STANISLAS Family Study, PIVUS study). Functional transcriptomic analyses were performed in peripheral blood mononuclear cells. Furthermore, in 403 healthy adults the associations between VEGF and adhesion/inflammation molecules were tested. Also, associations between VEGF and blood lipids were assessed in a discovery (n=1,006) and in a replication population (n=1,145) of healthy individuals.

Four polymorphisms (rs6921438, rs4416670, rs6993770, rs10738760) explaining ~50% of VEGF heritability were identified. These variants had significant effects on HDL, LDL, TNF-a, IL-6, E selectin and ICAM-1 plasma levels. rs6993770 was shown to increase VEGF121 mRNA levels and rs4416670 was associated with L-selectin expression. Common expression profiles were observed between VEGF, ICAM-1, L-selectin and TNF-a. Finally, significant associations were found between VEGF plasma levels and adhesion molecules in healthy adults.

Our integrative strategy resulted to significant results indicating molecular links between VEGF and cardiovascular disease biology and the importance of epistatic and gene x environment interactions. This example illustrates an improved strategy to be applied for every biomarker with high heritability levels using familial design and the existing biobanks

The investigation continues with pharmacogenomic studies of VEGF

In conclusion, new genetic variants in relation to VEGF, adhesion/inflammation molecules and blood lipids were identified, which can be targets for future pharmacogenomics studies that could assist in the prediction of better response to anti-VEGF therapies.

Potential to enhance colorectal cancer patient pathways through multiplex mutational profiling of KRAS, BRAF and PIK3CA genes

H.A. Murray1, J.E. Doherty1, M.P. Beaney1, M.J. Latten1, M.A. Crockard1, J.V. Lamont1, S.P. FitzGerald1

1Randox Laboratories Ltd, Crumlin, Northern Ireland

Background: Colorectal cancer is the third most common cancer worldwide. Despite recent therapeutic advances, the prognosis for patients with metastatic CRC (mCRC) remains poor. Monoclonal antibodies (moAb) targeting the epidermal growth factor receptor (EGFR) have proven to be effective in combination with chemotherapy or as single agents for the treatment of mCRC, however, patients harbouring KRAS mutations show resistance to anti-EGFR therapy. In addition up to 65% of patients with KRAS wild type tumours do not respond. Recent research suggests that mutations in other downstream effectors of the EGFR signally pathway, including BRAF and PIK3CA may also have a negative effect on patient response to anti-EGFR antibodies. We therefore report the analytical performance of an Evidence Investigator biochip array for the rapid simultaneous detection of 20 mutations within KRAS (including codons 12, 13, 61 and 146), BRAF (codon 600) and PIK3CA (codons 542, 545 and 1047) genes in CRC tissue samples.

Methods: The KRAS/BRAF/PIK3CA Array was used for the simultaneous qualitative detection of mutations within these genes from tissue (fresh, frozen or fixed [formalin fixed paraffin embedded (FFPE]) derived DNA. The assay is based on a combination of multiplex PCR and biochip array hybridisation. Innovative PCR priming technology permits high discrimination between multiple wild type and mutant DNA regions. Providing there are enough copies of DNA present, approximately 1% of mutant can readily be detected in a background of wild type genomic DNA. Target regions are limited to ≤100bp; permitting amplification of FFPE derived DNA, which could potentially be degraded. Analysis can be completed, from template DNA, through PCR (single reaction) to data readout in less than 3hrs. Dedicated software processes results automatically. Besides the 20 mutations detectable the assay also includes three internal positive controls for each of the genes providing evidence of correct performance of the array. Confirmation of array generated data was performed using several comparative methods such as bi-directional Sanger sequencing and pyrosequencing.

Results: Mutations across all three genes were readily detected using the KRAS/BRAF/PIK3CA Array in all tissue types analysed. 100% concordance with comparative methodologies was also achieved.

Conclusions: Results obtained demonstrate the applicability of KRAS/BRAF/PIK3CA Array to simultaneously detect mutations within these genes in CRC tissue samples, with high sensitivity and specificity. Detection of additional markers besides KRAS and extending the profile of this gene to include other codons beyond 12 and 13 may aid in the selection of candidate patients to receive anti-EGFR therapy, thereby maximising drug efficacy and minimising adverse patient effects.

AFFYMETRIX LUNCH SYMPOSIUM

Right drug, right patient, right dose, right time: The translation of pharmacogenetics into practice

Chair: Johanne McGregor, Ph.D., European Translational Research Marketing Manager, Affymetrix UK Ltd, UK

Today, we are facing a paradigm shift in the way we manage and treat patients with complex diseases. The pharmaceutical industry is facing unsustainably high attrition rates of compounds, mainly due to the lack of efficacy or demonstrated high toxicity at late stages of drug development. In addition, and importantly, the days of the “blockbuster drug” and treatment selection based on trial-and-error are rapidly coming to an end.

This represents a unique opportunity for pharmacogenomic research and its application in the clinic to make a considerable impact on patient care. Understanding the variation in genes encoding for drug metabolism enzymes and drug transporters allows us to predict the effect of an individual’s genetic variation on metabolic capacity. This understanding takes us one step closer towards the vision of personalized medicine by helping to avoid adverse drug responses, increasing treatment efficacy and providing both improved healthcare outcomes as well as substantial economic benefits. Implementing pharmacogenomics in the clinic, however, is not without its challenges.

In this seminar, Dr. Jesse Swen, Dept. Clinical Pharmacy & Toxicology, Leiden University Medical Center, will discuss key considerations when implementing pharmacogenomic testing in the clinic, focussing on the development of pharmacogenomic guidelines by the D utch P harmacogenetics W orking G roup. One such consideration is to improve clinical acceptance of pharmagenomic information by providing the opportunity to gain experience with pharmacogenomics through conducting a large cohort study in outpatients with the Affymetrix® DMET™ Plus Solution. The seminar will also provide an overview of how the DMET Plus Solution enables rapid genetic analysis of the involvement of metabolic pathways in drug metabolism.

For Research Use Only. Not for use in diagnostic procedures.

Development of pharmacogenetic guidelines by the Dutch Pharmacogenetics Working Group

Speaker: Jesse J. Swen, PharmD, Ph.D., Assistant Professor of Pharmacogenetics, Dept. Clinical Pharmacy & Toxicology, Leiden University Medical Center, The Netherlands

After the completion of the Human Genome Project in 2003, pharmacogenomics has become a mainstay of biomedical research. Yet, despite the significant body of evidence supporting its usefulness, routine pharmacogenomic testing in the clinic is still limited and fails to meet the initial high expectations. Dr. Swen will identify and present key factors to consider when implementing pharmacogenomic testing in a clinical setting. A major challenge in the translation of pharmacogenomics to clinical practice is the lack of guidelines directing the use of a pharmacogenomic test result. Dr. Swen will provide an overview of the currently available and future guidelines by the Dutch Pharmacogenetics Working Group (DPWG). He will also outline the methodology of the DPWG guidelines, discuss the perspective from which the guidelines are created and explain how the DPWG guidelines have been distributed nationwide in the Netherlands by integrating them with systems for computerized medication surveillance and drug prescribing and dispensing.

FRIDAY SEPTEMBER 27th AFTERNOON

THIRD SESSION – TRANSPORTERS AND PHARMACOGENOMICS

Introduction

Peter Meier-Abt - Swiss Academy of Medical Sciences, Petersplatz 13, 4051 Basel, Switzerland

Transporters and Pharmacogenomics

Wolfgang Sadee - OSU Program in Pharmacogenomics. College of Medicine. The Ohio State University. Columbus, OH 43210, USA

Drug response and toxicity can vary dramatically from one individual to the other, in part as a result of genetic factors. Several genetic variants have already been incorporated into clinical biomarker tests, bu the vast majority of genetic variability remains hidden. Extensive published data and our own results indicate that regulatory variants predominate over those directly affecting the primary protein sequence. We have implemented a gene-by-gene approach to search for regulatory variants, using allelic RNA expression ratios as a main tool. With next generation sequencing of RNA (RNAseq of coding and non-coding RNAs), we now search for regulatory variants genome-wide, or apply massively parallel analysis of multiple genes (AmpliSeq). These tools are applied to study pharmacologically important genes, including those encoding membrane transporters (SLCs and ABC). A survey of transporter expression reveals a multitude of expression profiles in various tissues (1,2), and the presence of multiple RNA transcripts at each gene locus (such as splice variants) (2). Focusing on neurotransmitter transporters, we have reported on several frequent regulatory variants in the dopamine transporter DAT (3). As DAT interacts physically with the dopamine D2 receptor in the pre-synapse, and DRD2 also carries frequent regulatory variants (4), we have tested epistatic interactions among them, comparing deceased subjects who had been heavy cocaine abusers with controls. A combination of a DAT and a DRD variants indeed conveyed nearly 8-fold enhanced risk of cocaine associated death (5). Further studies have revealed further instances of regulatory factors in transporter genes that may have clinically relevant effects. Supported by NIH U01 GM092655.

  1. Huang Y et al. Membrane Transporters and Channels: Role of the Transportome in Cancer Chemosensitivity and Chemoresistance. Cancer Res, 64:4294-4301, 2004.

  2. Webb A et al. Expression of mRNA transcripts encoding membrane transporters detected with whole transcriptome sequencing of human brain and liver. Pharmacogen.Genom. in press (2013).

  3. Pinsonneault JK et al. Dopamine transporter gene variant affecting expression in human brain is associated with bipolar disorder. Neuropsychopharm. 8: 1644-1655 (2011).

  4. Zhang Yet al. Novel polymorphisms in human dopamine D2 receptor gene affect gene expression, splicing, and neuronal activity during working memory. Proc.Natl.Acad.Sci.USA. 104: 20552-20557 (2007).

  5. Sullivan D et al. Dopamine transporter DAT and receptor DRD2 variants affect risk of lethal cocaine abuse: A gene-gene-environment interaction. Translat.Psych. 3, e222 (2013). doi:10.1038/tp.2012.146

DNA methylation and impact on transporter PGx

Schwab Matthias*, Schaeffeler Elke

*University Hospital, Tuebingen. Auerbachstrasse 112 70376, Stuttgart, Germany

In addition to genetic variability, epigenomic factors like DNA-methylation contribute to interindividual differences in drug response, best studied in cancer patients. Generally, DNA methylation results in gene silencing by direct inhibition of binding of transcription factors or by recruitment of methylated DNA-binding proteins. Tissue specific DNA methylation alters the expression of ADME genes such as cytochrome P450 enzymes (e.g. CYP1A2 enzyme) but also of drug transporters like efflux and uptake transporters. For instance the expression of the drug uptake transporters OCT1 in human liver and OCT2 in human kidney is majorly determined by DNA-methylation. Moreover, it has been shown that DNA methylation of the hepatic SLC22A1 is associated with downregulation of SLC22A1 in hepatocellular carcinoma (HCC) which might be a novel biomarker for HCC diagnosis and prognosis. Moreover, targeting SLC22A1 transporter methylation by demethylating agents like decitabine may offer a novel strategy for anticancer therapy of HCC. The large-scale, systematic epigenomic equivalents of GWAS, termed epigenome-wide associations studies (EWAS), are promising tools to determine specific drug-related phenotypes attributable to interindividual epigenomic variation. Interestingly, studies in 50-year-old versus 3-year-old monozygotic twin pairs showed remarkable differences in DNA methylation but also in histone acetylation, confirming that epigenetic marks are age dependent. Age dependency may limit the general use of DNA methylation as a biomarker for diagnosis and/or therapeutic response. References: Meyer UA, Zanger UM, Schwab M. Omics and drug response. Annu Rev Pharmacol Toxicol. 2013;53:475-502. Schaeffeler E, Hellerbrand C, Nies AT, Winter S, Kruck S, Hofmann U, van der Kuip H, Zanger UM, Koepsell H, Schwab M. DNA methylation is associated with downregulation of the organic cation transporter OCT1 (SLC22A1) in human hepatocellular carcinoma. Genome Med. 2011 Dec 23;3(12):82. Nies AT, Niemi M, Burk O, Winter S, Zanger UM, Stieger B, Schwab M, Schaeffeler E. Genetics is a major determinant of expression of the human hepatic uptake transporter OATP1B1, but not of OATP1B3 and OATP2B1. Genome Med. 2013 Jan 11;5(1):1.

miRNA and its impact on ABC transporter-mediated drug resistance

Ingolf Cascorbi, Institute of Experimental and Clinical Pharmacology, Christian Albrechts University, Kiel, Germany

Chemoresistance of tumours is often reported to be due to over-expression of efflux-transporters or genetic alterations of signalling pathways. Also the local bioavailability of drugs acting in the central nervous system may be subject of variability due to ABC efflux transporters located at the blood brain barrier. We could demonstrate that failure to anticonvulsants was associated genetic variants in ABCC2 in children or adolescents suffering from epilepsy. Very recently we demonstrated some relationship of polymorphic ABCB1 with Alzheimer’s disease, as the gene product P-gp was earlier proven to be involved in the efflux of amyloid-beta proteins.

There is also increasing evidence that epigenetic modification contributes to the phenomenon of drug resistance. Despite alteration of DNA methylation or histone-acetylation, deregulated microRNA expression patterns of tumour cells have been identified to interfere with drug response. Attempts to modify the expression of selected microRNAs have partly led to intriguing improvements of chemotherapy response. Down-regulation of certain microRNAs could abolish the suppression of gene expression as exemplified by imatinib resistance of BCR/ABL-over-expressing K-562 cells through up-regulation of ABCG2 (BCRP). There is currently a clinical study ongoing in CML patients to proof the role of microRNAs as biomarkers of imatinib response.

Recently we could show that the induction of ABCC2 protein by rifampicin is counteracted by microRNA 379 in HepG2 cells, contribution to the explanation of discrepancies between mRNA and protein expression following external stimuli though PXR ligands. Interestingly this microRNA interferes differentially with ABCC2-haplotypes. This finding may explain tissue-dependent differences in ABCC2 pharmacogenetics. Moreover, first findings indicate a change of mRNA and microRNA expression pattern after Y-roux surgery in severe obesity patients. Concluding, genetic and epigenetic factors contribute to inter-and intraindividual variation of ABC-transporter expression.

Characterization and regulation of the expression of drug transporters in human skin

Marion Alriquet, Anaïs Boulai, Magali Kouidhi, Karine Sevin, Alexandre Gaborit, Pierre Comby, Bernard Ruty and Hanan Osman-Ponchet. Metabolism & Pharmacokinetics Unit, Preclinical Development, Galderma R & D, Sophia-Antipolis, France.

Background: Most identified drug transporters belong to ATP-binding Cassette (ABC) and Solute Carrier (SLC). It has been recently recognized that like metabolizing enzymes, drug transporters may play an important role in pharmacokinetics and drug exposure and may be involved in clinically relevant drug-drug interactions. Transporter-based interactions have been well documented in liver and kidney for systemic drugs. However very little is known about the role of cutaneous drug transporters in the disposition of topically applied drugs.

The aim of this work has been to characterize the expression profile of SLC and ABC transporters in human skin, to compare it to liver’s and kidney’s and to study regulation of SLC and ABC transporter gene expression.

Methods: Gene expression: Total RNA was isolated from fresh human skin samples. Gene expression of eleven SLC transporters and four ABC transporters was measured by TaqMan Real-time RT-PCR. The genes studied were SLCO1B1, SLCO1B3, SLC22A1, SLC22A2, SLC22A6, SLC22A8, SLC47A1, SLC47A2, SLCO3A1, SLCO4A1, SLCO2B1, ABCB1, ABCC1, ABCC2 and ABCG2. As the kidney and liver are responsible for most drug excretion, the same experiment was performed with fresh and cryopreserved human hepatocytes (pool of 26 donors) and with RNA isolated from human kidney (2 donors).

Skin organoculture: Fresh human skin samples from 3 different donors were used and maintained in organoculture in 6-well plates. Four 6-mm biopsies were placed in each well filled with skin Long Term Culture Medium (Biopredic; France). Skin samples were treated with 20 µM Rifampicin during 72 hours with mediu m being renewed every 24 hours. Untreated skin samples were used as control. The culture plates were kept in a cell culture incubator set at 37°C, 5% CO 2 and saturated hygrometry. At the end of treatment period gene expression of SLC and ABC transporters was measured as described above.

Results: The results showed that SLC as well as ABC transporters have a very specific expression profile in human skin. Among the fifteen genes studied, expression of SLCO4A1 (OATPE), SLC47A1 (MATE1) and ABCC1 (MRP1) is the most important in human skin. Expression of six SLC transporter genes was not detected in human skin. In addition, expression profile of SLC and ABC transporters in skin is very different compared to hepatocytes. Indeed, expression of SLCO4A1 and ABCC1 is 70 times and 15 times higher in human skin than in human hepatocytes, respectively. Moreover, treatment of human skin with rifampicin during 72 hours leads to down-regulation of the expression of SLC47A1 (MATE1), SLC47A2 (MATE2), and ABCC1 (MRP1). Results showing the involvement of ABC transporters in drug absorption in human skin will be presented and discussed.

Conclusion: Expression of SLC and ABC transporters was characterized in human skin. Expression profile of SLC and ABC transporter genes is different in human skin, hepatocytes and kidney. Change of expression level of some SLC and ABC transporters by Rifampicin indicates that in human skin some SLC and ABC transporters may contribute to drug-drug interactions with topically applied drugs.

Drug transporters and pharmacogenomics of raloxifene

Janja Marc, Tina Trdan Lusin, Aleš Mrhar, Jurij Trontelj, Barbara Ostanek - Faculty of Pharmacy, University of Ljubljana, Slovenia

Raloxifene, a selective estrogen receptor modulator, exhibits quite large and unexplained interindividual variability in pharmacokinetics and pharmacodynamics. In our study was to determine the role of organic-anion transporting polypeptides OATP1B1 and OATP1B3 and their genetic variants in the pharmacokinetics and pharmacodynamics of raloxifene. To test the role of OATP1B1 and OATP1B3 transporters on hepatic uptake of raloxifene and its metabolites an in vitro model of Chinese Hamster Ovary cells expressing OATP1B1 or OATP1B3 was employed. The influence of OATP1B1 and OATP1B3 genetic variants on in vivo pharmacokinetics and pharmacodynamics was evaluated in 53 osteoporotic postmenopausal women treated with raloxifene. Our in vitro results showed that raloxifene and two of the three metabolites, raloxifene-4′-β-glucuronide (M2) and raloxifene-6,4′-diglucuronide (M3), interact with OATP1B1 and OATP1B3. Higher M3 and total raloxifene serum concentrations in patients correlated with lower serum levels of bone resorption marker, serum C-terminal telopeptide fragments of type I collagen, indicating a higher antiresorptive effect of raloxifene. Higher concentrations of M2 correlated with higher increase of lumbar spine bone mineral density supporting the raloxifene vertebral fracture specific protection effect. Finally, raloxifene, M3 and total raloxifene serum concentrations were significantly higher in patients with SLCO1B1 c.388A > G polymorphism and *1b haplotype implicating a considerable genetic effect on pharmacokinetics and pharmacodynamics of raloxifene. These findings indicate that SLCO1B1 c.388A > G polymorphism could play an important role in pharmacokinetics and pharmacodynamics of raloxifene.

The role of drug transporter genes in response of breast carcinoma patients

Hlavac Viktor, Brynychova Veronika, Vaclavikova Radka, Ehrlichova Marie, Vrana David, Pecha Vaclav, Trnkova Marketa, Kodet Roman, Mrhalova Marcela, Kubackova Katerina, Gut Ivan, Soucek Pavel

Background and Aims: Worldwide, breast cancer is the most common cancer in women. Multidrug resistance (MDR) remains one of the main obstacles to successful cancer treatment. ATP-binding cassette (ABC) transporters can contribute to MDR via ATP-dependent drug efflux pumps, e.g. P-glycoprotein. The Solute Carrier (SLC) transporters are essential for successful treatment by delivering drugs to cells. The major goal of our study was to investigate associations between the expression of ABC/SLC transporters, prognosis and response of breast carcinoma patients.

Material and methods: Expression profile of all known 49 human ABC transporter genes was evaluated in post-treatment tumor tissue samples from 68 breast cancer patients treated by FAC, FEC and/or taxane-based neoadjuvant chemotherapy regimens. Six ABC transporters were then evaluated in an independent set of 100 pretreatment patients. Expression profile of 21 human SLC transporters was evaluated in 33 post-treatment and 50 unselected tumor tissue samples. Transcript levels were compared between tumor and control tissues and between groups of patients divided by clinical prognostic factors and therapy response.

Results: The majority of drug transporters have shown deregulation of expression in post-treatment tumors compared with non-neoplastic control tissues. ABCA2 /A3 /A7 /A12, ABCB2 /B3 /B8/ B9 /B10, ABCC1 /C4 /C5 /C10 /C11 /C12, ABCD1 /D3, ABCE1, ABCF1 /F2 /F3, ABCG1, SLC22A7 /19A1 /29A3 and SLC31A1 were significantly overexpressed in tumors. ABCA5 /A6 /A8 /A9 /A10, ABCB1 /B5 /B11, ABCC6 /C9, ABCD2 /D4, ABCG5 /G8, SLCO1A2, SLC22A3 /22A6 /22A11 and SLC47A2 were significantly decreased in tumors. Significant associations of intratumoral levels of ABCC1 and ABCC8 with grade and expression of hormonal receptors were found in both sets of patients. SLCO1A2 level was higher in tumors from patients with smaller (pT1) compared with larger (pT2-4) tumors. ABCA12, ABCA13, ABCD2 and SLCO1A2 levels were significantly associated with the response to neoadjuvant chemotherapy in post-treatment patients. Protein expression of ABCA12, ABCC8 and ABCD2 was observed by immunoblotting in breast carcinoma tissues for the first time.

Conclusion: ABCA12, ABCA13, ABCC1, ABCC8, ABCD2 and SLCO1A2 represent candidate genes potentially modifying progression and response to the chemotherapy in patients with breast carcinoma. Validity of these potential biomarkers should be evaluated in independent follow-up studies and underlying mechanisms remain to be identified. This work was supported by grants no.: IGA NT13679-4 and CSF P303/12/G163.

SLC19A1 G allele as a biomarker of methotrexate-related gastrointestinal toxicity in Portuguese rheumatoid arthritis patients

Lima Aurea, Bernardes Miguel, Azevedo Rita, Sousa Hugo, Costa Lucia, Ventura Francisco, Seabra Vitor, Medeiros Rui

Objectives: Methotrexate (MTX) is the cornerstone drug for rheumatoid arthritis (RA) treatment. However, unpredictable MTX-related gastrointestinal toxicity (MTX-GastroTox) may occur. Aim was to determine potential biomarkers to predict MTX-GastroTox in Portuguese RA patients.

Methods: Patients (n=233) with active RA treated with MTX were followed-up. The influence of solute carrier 19 family 1 gene (SLC19A1/RFC-1) G80A polymorphism (rs1051266), baseline and clinical variables associated with MTX-GastroTox were analyzed. MTX-GastroTox was determined if patients presented any gastrointestinal adverse drug reaction (ADR) MTX-related. SLC19A1G80A polymorphism was genotyped by PCR-RFPL. Mann-Whitney’s, t-student and Chi-square tests were used to analyze variables. Multivariate regression analysis (MRA) was performed to evaluate the impact of several variables on MTX-GastroTox.

Results: In univariate analysis (UnA), results demonstrated a statistical significant association between MTX-GastroTox with disease duration (p=0.039) and subcutaneous (SC) MTX administration route (p<0.001). Folic acid supplementation was associated with MTX-GastroTox protection (p<0.001). Results also showed that higher disease duration was associated with higher maximum MTX doses reached (p=0.043). Furthermore, in MRA for MTX-GastroTox, when adjusting disease duration and maximum MTX doses, no associations were found.

Regarding genetic variable, in UnA, G carriers (p=0.011) and heterozygous GA (p=0.015) had almost 3-fold higher risk for MTX-GastroTox than AA homozygous. In MRA, adjusted for age, gender, smoking status, disease duration, folic acid, MTX administration route and maximum MTX dose reached, results showed that G carriers had 7-fold higher risk for MTX-GastroTox than AA patients (p=0.004;OR=7.03;95%CI=1.852-26.674). Moreover, SC route still associated with risk (p<0.001;OR=6.20;95%CI=2.335-16.489) and folic acid with protection (p=0.017;OR=0.36;95%CI=0.160-0.833) for MTX-GastroTox. Discussion: Longer disease duration seems to be associated with MTX resistance, which leads to MTX ineffectiveness and, consequently, to the need of higher MTX doses, responsible for the toxicity. Association between SC route and MTX-GastroTox possibly is due to the higher MTX bioavailability that conduces to toxicity. Folic acid supplementation is related with non-MTX-GastroTox. This drug is crucial for intracellular folate pool maintenance, that if not conveniently restored, depletion leads to toxicity. Furthermore, folic acid interacts with MTX leading to decreases MTX reabsorption and reducing MTX systemic levels, which may contribute to MTX-GastroTox protection. Patients G carriers are more prone to develop gastrointestinal ADRs. G allele has been implicated in lower MTX transport activity by RFC-1, decreasing MTX intracellular levels with consequent increase MTX systemic levels, conducing to toxicity.

Conclusions: Optimal triad for MTX-GastroTox protection includes genotype AA for SLC19A1G80A polymorphism, folic acid supplementation and per os MTX administration route. Findings indicate that genotyping SLC19A1G80A polymorphism may help clinicians to identify patients whom will not benefit from MTX treatment due to potential occurrence of gastrointestinal ADRs.

The frequency of allele genotypes of gene SLCO1B1*5 in Russian patients suffering from hyperlipidemia

Sychev Dmitry, Shuev Grigory, Grachev Andrey, Beloshitskaya Tatyana

Introduction: SLCO1B1 gene encodes a polypeptide transporting organic anions and participating in the removal of statins by the liver into bile. It is now known that the variant allele carriers of SLCO1B1*5 is associated with a high risk of myopathy, rhabdomyolysis until, with statins: simvastatin, atorvastatin, pravastatin, rosuvastatin. Patients carriers (both heterozygous and homozygous) allelic variants SLCO1B1*5 myopathy with statins at high doses is found in 60% of cases.

Objective: To study the prevalence of genotypes of allelic variants of the gene SLCO1B1 * 5 patients among the Russian population in the Moscow region with hyperlipidemia, which is planned appointment of statins. Materials and methods: The study included 572 patients aged 58 ± 11, 235 men (41%) and 337 women (59%) who planned appointment of statins. All patients were genotyped for variant SLCO1B1 * 5 (s.521T> C, rs4149056) by Real-Time PCR after prior isolation of DNA from blood leukocytes.

Results and Discussion: As a result of genotyping by allelic variant SLCO1B1 * 5 of 572 patients had 349 TT genotype (61%), 186 - TC genotype (32.5%) and 37 - CC genotype (6.5%). Statistically significant differences from the Hardy-Weinberg equilibrium was not observed (X2 = 1,38 p = 0,24). In identifying the TT genotype genetically determined risk of myopathy was regarded as a low, TC - the average and the CC - as high. In this case applied the framework for selecting the maximum dose of statins during the titration, is guided by certain genotype SLCO1B1 * 5, on the basis of already existing recommendations.

Conclusions: The current definition of genotypes by allelic variant SLCO1B1 * 5, is recommended for the practice of the European Science Foundation (ESF). Moreover, this pharmacogenetic test is indicated for the prevention of development of myopathy (including rhabdomyolysis) in patients with hyperlipidemia who planned the use of statins and personalized choice of the maximum dose of statins. Our results show that among Russian patients with hyperlipidemia, which need statin therapy, often found CT and CC genotypes by allelic variant SLCO1B1 * 5, associated with medium to high risk of myopathy, respectively. We can assume that pharmacogenetic testing for personalization choice dosing regimens of statins may reduce the risk of statin-induced myopathy, but this must be proved in conducting prospective studies.

SATURDAY SEPTEMBER 28th MORNING

FOURTH SESSION – STEM CELLS AND OTHER NEW TOOLS FOR PHARMACOGENOMICS AND DRUG DISCOVERY

Introduction

Urs A. Meyer - Biozentrum, University of Basel, Switzerland

New and “old” in vitro tools for the study of polymorphic variants of the human cytochrome P450 enzyme complex

Michel Kranendonk - CIGMH/Department of Genetics, Faculty of Medical Sciences, Universidade Nova de Lisboa

Variation in efficacy and toxicity of pharmaceutical drugs are related to environmental and genetic factors which are involved both in pharmacokinetic as well as pharmacodymanic characteristics of these compounds. Genetic polymorphism in the ADME processes, in particular metabolism (biotransformation) has been described as one of the major causes of variation in drug-efficacy and adverse drug reactions (ADRs). Different in vitro approaches are in use to study the effect of genetic variations in human biotransformation allowing the delineation of metabolic pathways, in particular potential bioactivation routes, a major event in ADR. More than 70% of the currently most prescribed drugs are metabolized by the human cytochrome P450 enzymes of families 1-3. These P450 enzymes are highly polymorphic and their genetic variability has been considered to play a major role in the variation of drug therapy outcome. The detailed knowledge of the influence of this genetic variation on drug metabolism is therefore paramount in the improvement of drug efficacy and safety. The application of a formerly developed (“old”) cell system, for the study of genetic variants of human P450 enzymes will be presented by which not only insight was obtained on their role in variability in biotransformation as well on the functional, molecular mechanism of this enzyme complex. Furthermore, the development of a new, human P450 competent cell model for use in a high through-put bioactivation assay will be presented. Its application for the study of the role of P450 genetic variability in the generation of reactive metabolites will be discussed, a causal event in drug induced liver injury (DILI) and major reason for impediment of drug development and clinical use after marketing.

REFERENCES

Kranendonk et al., (1999) Mutat Res 441: 73-83.

Moutinho et al., (2012), Drug Metabolism & Disposition 40: 754-60

Palma et al., (2013), Pharmacogenetics and Genomics, 23: 41-52

ACKNOWLEDGEMENTS: supported by NIH (grant GM081568) and FCT (grant PTDC/SAU-GMG/71911/2006)

Stem cells and their progenies as a novel tool for drug screening

Lino Ferreira - Center for Neurosciences and Cell Biology, University of Coimbra

In the first part of my talk, I will present our results in the generation of an in vitro human BBB model. The human blood brain barrier (BBB) is a selective barrier formed by brain endothelial cells (hBECs), which is important to ensure adequate neuronal function and protect the central nervous system (CNS) from disease. The development of human in vitro BBB models is thus of utmost importance for drug discovery programs related to CNS diseases. I will present a method to generate a human BBB model using cord blood-derived hematopoietic stem cells. The cells were initially differentiated into ECs followed by the induction of BBB properties by co-culture with pericytes. The brain-like endothelial cells (BLECs) express tight junctions and transporters typically observed in brain endothelium and maintain expression of most in vivo BBB properties for at least 20 days. The model is very reproducible and can be generated from stem cells isolated from different donors and in different laboratories. We provide evidence that Wnt/β-catenin signaling pathway mediates in part the BBB inductive properties of pericytes. In the second part of my talk, I will present recent results about the use of stem cells to generate vascular models for toxicity and drug screening analyses. We demonstrate the utility of embryonic arterial ECs cultured under flow conditions for toxicological assessment.

SSRI response biomarkers: lessons from genome-wide transcriptomic profiling of human lymphoblastoid cell lines

David Gurwitz, Department of Human Molecular Genetics and Biochemistry, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 69978, Israel Email: gurwitz@post.tau.ac.il

Genome-wide pharmacogenomic studies for developing targeted therapies offer the advantage of hypothesis-free search for tentative drug response biomarkers (efficacy and safety). However, they require large patient cohorts and are therefore laborious and quite costly. Here we present our experience with an alternative approach, based on genome-wide transcriptomic profiling of a panel of human lymphoblastoid cell lines (LCLs) representing unrelated healthy donors. LCLs can be obtained from most large biobanks and our approach offer simple and inexpensive discovery of tentative drug response biomarkers. The expression of candidate biomarker genes and microRNAs (miRNAs) is subsequently measured by qPCR in blood samples of patient cohorts.

We applied genome-wide expression profiling of human LCLs for searching tentative SSRI antidepressant drug response biomarkers. Eighty LCLs from healthy adult female individuals were phenotyped for growth inhibition by paroxetine. Fourteen LCLs were chosen for comparative expression profiling with Affymetrix microarrays. The most notable difference between LCLs displaying high vs. low paroxetine sensitivities was a 6.3-fold lower basal expression (p=0.0000256) of CHL1 (close homologue of L1), coding for neuronal cell adhesion protein implicated in thalamo-cortical circuitry. This was confirmed by qPCR.

Next, our studies with commercial miRNA arrays have identified miR-151-3p, predicted to target CHL1, as an additional biomarker for SSRI sensitivity. This finding supports the role of CHL1 expression levels in predicting SSRI sensitivity. Other miRNAs showing differential expression levels in the two groups of human LCLs, which corresponds with the differential expression of other tentative biomarker genes, included miR-132, miR-212, miR-30b* (miR-30b-3p), let-7b and let-7c, all of which are also implicated in CNS function.

Preliminary findings will be presented from our ongoing studies with DNA samples from major depression patients with known SSRI response phenotypes.

Our studies demonstrate that drug sensitivity phenotyping in LCLs from unrelated healthy donors, followed by their genome-wide expression profiling for genes and miRNAs, is a powerful and cost-effective tool for searching drug response biomarkers (prior to collection of clinical samples). The method is applicable for drugs, including CNS drugs, whose targets are functionally expressed by LCLs. This approach builds upon the utility of LCLs of healthy donors to faithfully represent the intricate human transcriptomic repertoire.

The “Virtual Pancreatic Beta-Cell”: A Modeling and Simulation Approach in the Development of Anti-Diabetic Treatments

Hans-Christoph Schneider - Sanofi-Aventis Deutschland GmbH, R&D LGCR/Structure, Design & Informatics, Computational Biology & Bioinformatics, 65926 Frankfurt, Germany

Modeling and simulation approaches aim to build a representation of biochemical and physiological processes in the form of dynamic mathematical models. These models then are used to run simulations of “virtual” experimental layouts on a computer. The results give guidance for various aspects of the research process. Here, we describe the development of a mathematical model for the support of diabetes drug discovery.

Pancreatic beta cells secrete the hormone insulin in response to increasing blood glucose levels and are a major focus of diabetes research. So far, only isolated aspects of glucose stimulated insulin secretion (GSIS) and its amplification by receptor activated pathways have been captured in mathematical models. A comprehensive model that integrates the complete sequence of events in a single model has not been published so far.

We have selected three published ODE-based models on individual aspects of GSIS and used them as modules to construct a comprehensive “virtual pancreatic beta-cell”. These components were joined by adaptors containing simple mathematical functions. The complete model was fitted to reproduce the experimental model of the isolated perfused rat pancreas.

For validation of the “virtual pancreatic beta-cell” we simulated the effect of two important classes of anti-diabetic drugs, namely the K-ATP-channel inhibitors (sulfonylureas) and the GLP1-receptor agonists. These simulations were able to reproduce all major aspects of these drugs found in corresponding experiments. A sensitivity analysis resulted in high sensitivities for components that are known to have a strong effect on insulin secretion, e.g. glucokinase and voltage gated potassium channels. This result further validates the model.

In conclusion, we have built a comprehensive ODE based mathematical model of the pancreatic beta-cell. This model is able to simulate the effects of existing or potential pharmacological interventions on insulin secretion, and therefore presents a powerful tool in the development of anti-diabetic treatments.

APOE-TOMM40 and CYP2D6 in the pharmacogenomics of dementia

Ramón Cacabelos - Chair of Genomic Medicine, Camilo José Cela University, Madrid, Spain

Dementia is a major problem of health in developed countries. About 10% of direct costs of dementia are attributed to pharmacological treatment, with poor cost-effectiveness. Different studies indicate that the therapeutic outcome in Alzheimer’s disease (AD) is genotype-specific. Pharmacogenomics may account for 50-80% variability in pharmacokinetics and pharmacodynamics. The genes involved in the pharmacogenomic response to drugs in AD fall into five major categories: (i) genes associated with AD pathogenesis and neurodegeneration (APP, PSEN1, PSEN2, MAPT, PRNP, APOE and others); (ii) genes associated with the mechanism of action of drugs (enzymes, receptors, transmitters, messengers); (iii) genes associated with drug metabolism (phase I (CYPs) and phase II reactions (UGTs, NATs)); (iv) genes associated with drug transporters (ABCs, SLCs); and (v) pleiotropic genes involved in multifaceted cascades and metabolic reactions (APOs, ILs, MTHFR, ACE, AGT, NOS, etc). Multiple polymorphic risk variants can increase neuronal vulnerability to premature death. Among these susceptibility genes, the apolipoprotein E (APOE) gene (19q13.2)(AD2) is the most prevalent as a risk factor for AD, especially in those subjects harboring the APOE-4 allele, whereas carriers of the APOE-2 allele might be protected against dementia. APOE-related pathogenic mechanisms are also associated with brain aging and with the neuropathological hallmarks of AD. The APOE-TOMM40 cluster and CYP2D6 influence the therapeutic response to conventional drugs in AD. The response rate by genotype is as follows: APOE-2/3: 44.26% responders (RRs), 36.07% non-responders (NRs), 19.67% stable-responders (SRs); APOE-2/4: 55.56% RRs, 44.44% NRs, 0.0% SRs; APOE-3/3: 63.42% RRs, 21.06% NRs, 15.52% SRs; APOE-3/4: 56.94% RRs, 27.75% NRs, 15.31% SRs; APOE-4/4: 51.43% RRs, 28.57% NRs, 20.00% SRs. CYP2D6-EM are the best responders and PMs and UMs are the worst responders. In addition, APOE-4/4 carriers tend to accumulate in CYP2D6-PMs and UMs. The introduction of pharmacogenomic procedures into AD therapeutics may contribute to optimizing the pharmacological treatment of dementia.

SPECIFIC PHARMACOGENOMICS PRESENTATIONS

Sulfolane levels and its relation to CYP2C9 genotypes in children receiving intravenous busulfan prior to hematopoietic stem cell transplantation

Uppugunduri Chakradhara Rao S, Daali Youssef, Rezgui Mohamed Aziz, Huezo Diaz Patricia, Tyagi Anuj Kumar, Rousseau Julie, Duval Michel, Bittencourt Henrique, Krajinovic Maja, Ansari Marc

Cytochrome P 450 enzymes (CYPs) were presumed to play a role in the oxidation of intermediate metabolites of busulfan (Bu). In vitro elucidation of involvement of CYPs in the oxidation of Bu metabolites is cumbersome due to the volatile nature of tetrahydrothiophene and non-availability of sensitive quantitation methods. This study is aimed at exploring the association of CYP2C9, CYP2C19, CYP2B6, FMO genotypes and sulfolane (Su) levels in children undergoing hematopoietic stem cell transplantation (HSCT). The relation of genotypes with the outcomes of HSCT was also explored. Sixty six children receiving intravenous Bu based myeloablative conditioning regimen were genotyped for common functional variant alleles in CYP2C9 (*2 and *3), CYP2C19 (*2 and *17), FMO3 (rs2266780, rs2266782 and rs1736557) and CYP2B6 (*5 and *9). Institute review board approved the study, and samples were collected only after obtaining informed consent from the patients or parents. Genomic DNA extracted form whole blood collected before conditioning was used for whole genome amplification (WGA). This WGA DNA was utilized for genotyping using TaqMan® Drug Metabolism Genotyping Assays on a StepOnePlus™ Real-Time PCR System. Plasma levels of Bu and its metabolite Su were measured after dose 9 from a subset of 44 patients for whom plasma samples after dose 9 were available using dried plasma spotting followed by LC-MS/MS analysis for Bu and GC-MS/MS analysis for Su. The ratio of Bu to Su was taken as a metabolic ratio (MR) to compare among genotype groups. The MRs (Bu/Su), Bu and Su levels between different genotype groups were compared using non-parametric tests. Cumulative incidence of overall survival (OS) and event-free survival (EFS) were estimated using Kaplan-Meier curves and log-rank test was used to compare the difference between genotype groups or groups divided on the basis of MR, in a univariate analysis. Multivariate analysis for cumulative incidence of EFS and OS was performed using cox-regression analysis. Higher metabolic ratios (MRs, Bu/Su) were observed in CYP2C9*2 and *3 allele carriers (mean±SD, 7.8±3.6 Vs 4.4±2.2; p=0.003). Lower event-free survival was seen in patients with MR above the median 5 (40 Vs 79%, p=0.009) and carrying reduced function alleles of CYP2B6 (40 Vs 84 %, p=0.005). In multivariate analysis non-malignant disease status (82.6 % Vs 48.8 % in malignant patients), MR below 5 and CYP2B6 normal haplotypes were independently associated with better EFS (p<0.05). This study suggests the role of CYP2C9 in the oxidation reactions of THT and CYP genotypes along with Bu MRs to be important at predicting outcomes of Bu based myeloablative conditioning prior to HSCT. MR of Bu after dose 9 infusion provides an idea about CYP2C9 activity and possibly the extent of cyclophosphamide activation. Finally, association of lower EFS with CYP2B6 loss of function variants and higher Bu MRs could be used clinically to predict EFS.

CYP2W1: Endogenous function and role in cancer therapy

Choong Eva, Guo Jia, Persson Anna, Virding Susanne, Mkrtchian Souren, Ingelman-Sundberg Magnus

Colorectal cancer (CRC) is the third most common cancer worldwide. We have recently shown CRC-specific expression of the orphan cytochrome P450 2W1 (CYP2W1) that belongs to a P450 monoxygenases family involved in detoxification of xenobiotics, drugs and also in the metabolism of certain endogenous compounds. CYP2W1 expression is associated with less survival rate in CRC stages II and III (1, 2). CYP2W1 was suggested as an attractive target for CRC therapy by exploiting its ability to activate certain prodrugs to cytotoxic metabolites. This hypothesis was successfully confirmed in our laboratory both in vitro and in vivo on a murine xenograft model (3). In this context, the exploration of the mechanisms that control the CYP2W1 expression is of paramount importance as it may substantially increase the number of patients who would benefit from such CRC-specific therapy, including firstly the induction of CYP2W1 (in case of low or absent expression), and secondly treatment with CYP2W1-specific prodrugs. The key for determining CYP2W1 regulation might be found by studying the developmental pattern of its expression. The cyp2w1 protein has been detected in rat fetal colon and the gene is subsequently silenced in adult age (4), the expression pattern is reminiscent of the expression of many oncofetal genes. In this report, we present data on the analysis of the expression of cyp2w1 in mice colorectal tissues oof different ages from fetus to adulthood. Moreover, we have examined whether the same principle of specific fetal colon expression applies also to humans by analyzing the CYP2W1 expression on the mRNA and protein levels in fetal human colon (obtained from the NICHD Brain and Tissue Bank for Developmental Disorders at the University of Maryland, Baltimore, MD (USA)). In addition, we also present data on the CYP2W1 regulatory mechanisms, potential substrates, inductors and inhibitors investigated in the different cell lines expressing CYP2W1. In conclusion, we examined the mechanisms of regulation and induction of the CYP2W1 expression. The project could provide novel information of high relevance for future treatment of colon cancer. Note: This study was approved by the Ethical Committee at Karolinska Institutet, Stockholm (Sweden). Literature 1. D. Edler et al., Eur J Cancer (2009). 2. K. Stenstedt et al., Anticancer research (2012). 3. S. Travica et al., Clin Cancer Res (2013). 4. M. Karlgren et al., Biochemical and biophysical research communications (2006).

Inclusion of genetic information in population pharmacokinetic models

Tessier Adrien, Bertrand Julie, Fouliard Sylvain, Chenel Marylore, Comets Emmanuelle

Objective: In pharmacokinetic (PK)/pharmacodynamic (PD) studies, a population approach, using non-linear mixed effect models (NLMEM), is frequently used to identify the sources of variability in drug response, such as genetic profile. Recent advances of genotyping techniques, such as DNA microarray, allow identifying simultaneously many genetic markers, generally Single Nucleotide Polymorphisms (SNP). However, there is no consensus on how to integrate such a large number of polymorphisms in population models. We propose to identify and evaluate methods to study the impact of SNPs on the parameters of NLMEM.

Methods: In population PK/PD, covariate model building is generally performed using a stepwise procedure, with covariates included one by one after an initial screening step based on univariate analyses [1]. Lehr proposed an algorithm based on this procedure adapted to genetic covariates by testing the correlation between candidate covariates [2]. An alternative is to analyse all markers simultaneously using a penalization function to force the less significant coefficients towards zero, selecting a parsimonious covariate model. We identified three such methods: Ridge regression, adapted to include a test of significance for its derived estimates [3]; Lasso [4]; HyperLasso [5], a generalization of the Lasso. We compared these four methods through a simulation study, using data from drug S, currently in development in SERVIER laboratories. The dataset includes data of three Phase I studies including 78 subjects; 176 SNPs of 39 genes were genotyped using specific SERVIER DNA microarray, chosen for their known implication in drugs pharmacokinetics. 200 datasets were simulated for two scenarios using a model describing the complex PK of drug S. In the first scenario (H0), no SNP had an effect on the PK. In the second (H1), we assumed that 6 SNPs in each simulated dataset had an impact on the elimination clearance. The value of the regression coefficients for these 6 SNPs were chosen to explain a total of 30% of the interindividual variability of clearance. Evaluation was based on the performance of each method in terms of false and true positive rates. Then, the four methods were applied to study the impact of the SNPs on clearance.

Results: The drug S pharmacokinetics was best described by a two compartments model, with a zero order absorption and a late absorption used to fit the rebound in concentrations observed at 24 hours. A preliminary application of the four described methods on real data set highlighted the impact of several SNPs on individual clearances, one of which is picked up by all methods and corresponds to a marker of metabolic enzyme NAT1.

Discussion: Exploration of DNA microarray is confronted with: the large number of genetic covariates compared to the number of observations, the correlation between these covariates and the fact that variability could be explained by the addition of several variants with moderate effect rather than by the large effect of a single variant. Here genetic statistic methods manage these different issues and NLMEM allow better understanding of the impact and mechanism of genetic markers on primary PK parameters.

The impact of FSH receptor gene polymorphism 2039G>A on controlled ovarian stimulation response in women undergoing in vitro fertilization

Dumic Jerka, Renato Bauman, Sanja Dabelic, Sandra Supraha Goreta

Single nucleotide polymorphism (SNP) 2039G>A (Asn680Ser) in the follicle stimulating hormone receptor gene (FSHR) has been related to the women’s response to the controlled ovarian stimulation (COS), which is one of the key factors for the successful in vitro fertilization. To assess the potential of this SNP for predicting ovarian response to FSH stimulation, 211 women undergoing IVF were genotyped. In addition to the hormonal status determination (the basal concentrations of FSH, luteinizing hormone (LH), estradiol (E2), thyroid-stimulating hormone (TSH) and prolactin), the number of antral follicles was estimated, as well. It was found that genotype 2039G/A (Asn680/Ser680) of FSHR is neither a good predictor of ovarian reserve nor of ovarian response upon controlled FSH stimulation. Consequently, the genotyping of SNP 2039G>A of FSHR for identifying poor FSH responders following controlled ovarian stimulation (COS) is not diagnostically valuable. Yet, women of younger age (< 32 years) (63 women), who were carriers of the homozygous form (2039A/A, Ser680/Ser680) (16 women), previously related to the poor ovarian response, had significantly higher number of oocytes (comparing to other genotypes of the same age, and also to the same genotype of different age; p < 0.01). Consequently, they were recognized as predisposed for hyper-response upon FSH controlled stimulation. Thus, genotyping for this FSHR SNP 2039G/A seems to be a promising biomarker for pre-identifying women who should be treated with lower FSH dosage during IVF. Further studies are required to assess the role of this polymorphism, including additional alleles, in FSH hypersensitivity reactions in women undergoing IVF procedures.

SATURDAY SEPTEMBER 28th AFTERNOON

FIFTH SESSION – FROM SYSTEM PHARMACOGENOMICS TO PERSONALIZED MEDICINE

Personalized Medicine and Pharmacogenomics

Angela Brand

European Experiences of Pharmacogenomics

Krishna Prasad

Modelisation in Pharmacology

Helder Mota Filipe

Network modeling to identify new mechanism and therapeutic targets for Parkinson’s disease

Howard J Federoff, Habtom Ressom, Salim Shah and Linda MacArthur - Georgetown University Medical Center, Washington DC, USA

Biomolecules in subnetworks are the focus of a new strategy to develop drugs that halt complex diseases. In this article, we use Genome Wide Association Study (GWAS) and linkage data derived from Parkinson’s disease studies to illustrate how algorithms that use gene and protein interaction databases can reveal subnetworks in biological systems that suggest mechanisms for disease progression. Further, network modeling may help develop testable hypotheses for neurodegenerative diseases and open up new avenues for therapeutic development.

European Commission representative

Christina Kyriakopoulou (Scientific officer, Health Directorate, DG General for Research & Innovation Brussels, Belgium) – to be confirmed

Personalized Medicine for the European Citizen

Kirsten Steinhausen

A foresight exercise was undertaken by the European Science Foundation to identify the main challenges in the development and implementation of personalised medicine across Europe. A multi- and interdisciplinary group of key stakeholders, including patient groups, regulators, industry and academics, were consulted and invited to discuss and propose recommendations related to personalised medicine in four core areas: data handling; the introduction of new models and decision-making processes (from safety assessment to reimbursement procedures); the enhancement of interdisciplinarity and stakeholder participation; and the need for new infrastructure and funding resources. The results of these discussions were published in December 2013 the Report “Personalised Medicine for the European Citizen: Towards more precise medicine for the diagnosis, treatment and prevention of disease (iPM).

The report resulted in 80 recommendations, which were condensed into six core recommendations applied to a circle model that represents the target for personalised medicine.

The implementation of personalised medicine necessitates a close collaboration and engagement of all stakeholders and the involvement of governments at national and European level. Further discussion and work is needed to transform personalised medicine to personalised healthcare for the benefit of all European citizens.

Poster abtracts

Correlation between inhibitor developement and F8 mutation in hemophilia A patients from west Algeria

Abdi Meriem, Zemani Fodil Faouzia, Fodil Mostefa, Boudjema Abdallah, Saidi-Mehtar Nadhira

One of the most important predictors of the risk of inhibitor development in severe hemophilia A is the factor 8 gene mutation type. Our main objective was first to detect presence of inhibitor in 24 HA patient from West Algeria. Then, we detected the mutation in the factor 8 gene in these patients. FVIII inhibitor titration was performed according to Bethesda/Nijmegen modification assays. Intron 22 and intron 1 inversions were respectively detected using Long Rang Polymerase Chain Reaction and Multiplex Polymerase Chain Reaction. Patients who were negative for both inversions were analyzed using a direct sequencing. Inhibitors were found in only 12.5% of the patients with severe form of HA. From these patients, one with nonsense mutation (c.322A>T, p.Lys108*), one with the polymorphism (c.3780C>G, p.Asp1260Glu) and one with intron 22 inversion. However, only one patient developed inhibitor from intron 22 inversion sub-group, which indicates implication of other factors in inhibitor development. Further analyses are required in order to determine genetic predictors of inhibitor development in our population

Pharmacogenomic landscape of the Lithuanian population

Aidas Pranculis, Vaidutis Kučinskas

The LITGEN project was launched in 2011 with the aim of determining the genetic diversity and structure of the population of Lithuania using informative variable pharmacogenetic markers including SNPs, CNVs, STRs of Y chromosome, mtDNA, autosomes, exome and whole genome for future extensive wide scale human genome studies in Lithuania. As part of the project the efforts were launched to use the data obtained in order to identify the pharmacogenetic peculiarities of the Lithuanian population. Currently there are DNA samples obtained from over 1000 healthy unrelated individuals from various ethno-linguistic regions of Lithuania. All of the participants of the study signed informed consent forms, filled out lifestyle and nutrition questionnaires and had their biochemical phenotype associated with cardiovascular diseases determined. Large scale genotyping is performed using Illumina HumanOmniExpress-12 v1.0 genotyping array on the Illumina HiScanSQ system. The determined pharmacogenes of particular interest and imporance to the Lithuanian population will be analyzed through exome and whole genome sequencing on the Life Technologies SOLiD 5500 system. The analysis was started by genotyping 96 individuals with HumanOmniExpress-12 v1.0 genotyping arrays. Particular attention was devoted to the analysis of SNPs and CNVs within the genes important for the treatment of cardiovascular disease. Preliminary results revealed that based on allele frequency distribution of the VKORC1 c.*134G>A polymorphism compared to patients from other Caucasian populations, Lithuanian patients may require higher doses of warfarin to achieve the same therapeutic effect and that they are in some cases twice as likely to develop ADRs when treated with statin class drugs, because of the frequency of the risk C allele in the c.521T>C polymorphism of the SLCO1B1 gene. Further analysis of the PTGS2 -765G>C revealed a significantly lower C allele frequency (p<0.03) in subjects from Lithuanian population compared to other Caucasian populations indicating that higher aspirin doses may be beneficial to patients in order to achieve desired antithrombotic results. Currently further genotyping is being carried out to replicate and confirm the results in a larger study group and to select genes and genomic regions for sequencing.

LITGEN project (VP1-3.1-ŠMM-07-K-01-013) is funded by Global Grant.

Influence of CYP3A4 genotypes in the outcome of serous ovarian cancer patients treated with first-line chemotherapy: definition of a CYP3A4 activity profile

Assis Joana, Pereira Deolinda, Gomes Mónica, Marques Dânia, Marques Inês, Nogueira Augusto, Catarino Raquel, Medeiros Rui

Background Ovarian cancer (OC) is one of the most common causes of cancer-related death among women and the major cause of death due to gynecological cancer. Standard treatment for OC patients is based on cytoreductive surgery, followed by first-line chemotherapy with platinum (cisplatin or carboplatin) and taxane agents (paclitaxel or docetaxel). However, despite this aggressive approach, the 5-year survival rates remains only around 45 %. CYP3A4 is a key enzyme involved in the metabolism of numerous compounds, such as paclitaxel, as its activity shows an extensive inter-individual variation which can influence treatment response. The aim of the study was to evaluate the potential predictive role of a CYP3A4 profile (rs2740574 and rs35599367) in ovarian cancer patients treated with first-line chemotherapy. Material and Methods To undertake this work, a group of patients with ovarian cancer was selected (n=261). We analyzed CYP3A4 polymorphisms in genomic DNA of patients with serous ovarian cancer treated with first-line chemotherapy (paclitaxel and cisplatin or carboplatin), after cytoreductive surgery. CYP3A4*1B and CYP3A4*22 genotypes were determined by Nested PCR-RFLP and Taqman® Allelic Discrimination, respectively.

Results We observed that the mean survival rates were statistically different according the patients CYP3A4 genotypes. The group of patients carrying the CYP3A4*1B G allele present a decreased mean survival rate when compared with AA genotype patients (103,93 and 134,44 months, respectively, p=0,010). This result is consistent after multivariate Cox regression analysis [HR, 2,15; 95 % CI, 1,03-4,52; p=0,043]. The combination of CYP3A4*1B and CYP3A4*22 polymorphisms results in the definition of a CYP3A4 activity profile: the group of patients with a higher CYP3A4 activity profile had significantly diminished survival when compared with patients with a lower CYP3A4 activity profile (101,06 and 134,44 months, respectively, p=0,012). Multivariate Cox regression analysis revealed a diminished overall survival time for patients with CYP3A4 high activity profile (HR, 2,29; 95 % CI, 1,05-5,02; p=0,038). The definition of a CYP3A4 activity profile resulted in the increase of prediction ability, using Harrels’s concordance indexes (C-index from 0,617 to 0,626).

Conclusion To the best of our knowledge this is the first study that evaluate the presence of CYP3A4*1B polymorphism in the overall survival of OC patients and the first study to evaluate the influence of the new genetic CYP3A4 intron 6 polymorphism in OC patients. Our results demonstrate an association between CYP3A4*1B and a diminished overall survival of patients with serous ovarian cancer. The definition of a CYP3A4 activity profile proved to be benefic and the CYP3A4 high activity profile was associated with a lower overall survival. We consider that the definition of a CYP3A4 activity profile might be useful as molecular marker for predicting the clinical outcome of OC patients.

A tendency toward higher plasma efavirenz concentration in carriers of the ABCB1 c.3435 C>T polymorphism

Barusrux Sahapat, Koomdee Napatrupron, Santon Siwalee, Jantararoungtong Thawinee, Prommas Santirat, Chamnanphol Montri, Puangpetch Apichaya, Sukasem Chonlaphat

Aim This study was designed to examined the frequency of ABCB1 c.3435 C>T polymorphism including the impact on efavirenz plasma concentration in HIV-1 infected Thai patients receiving efavirenz. Methods: c.3435 C>T (rs 1045642) polymorphism at the gene encoding the ABCB1 in 149 HIV-infected Thai adults were genotyped. Plasma efavirenz concentrations at 12 hours after dosing were measured using a validated high performance liquid chromatopraphy. Relationship between plasma efavirenz concentrations and c.3435 C>T polymorphims were analysed.

Results The frequency of c.3435 C>T heterozygous (C/T) and homozygous mutant (T/T) was 53.02% and 18.12%, respectively (Minor allele frequency= 0.45). In the entire cohort, the median efavirenz plasma concentration was 2,410 ng/mL (IQR 1,460-4,120). No significant association with ABCB1 c.3435 C>T polymorphims in Thai HIV-infected patinets. Median plasma efavirenz concentration for patients with c.3435 TT (2,730 ng/mL, IQR 2,020-4,190) and CT (2,290 ng/mL, IQR 1,410-4,280) genotype were not significant higher than those with homozygous wild-type (2,100 ng/mL, IQR 1,370-3,530).

Conclusions A tendency toward higher efavirenz concentration in carriers of the ABCB1 c.3435 C>T polymorphism was found in this study, but this difference was not significant. Study with large HIV-infected patients might be needed to demonstrate an association of ABCB1 gene and efavirenz pharmacokinetics.

Supervision of Fungal infections, Aspergillosis, at the patients at high risk. In CHU of Oran

Benmansour Zakaria

Introduction The presence of molds in a hospital environment became a subject of concern both for the healthcare professionals and for the users. Indeed, in spite of the absence of indicators allowing to measure their roles in the arisen of the fungal infections, it is established that bio contamination at the hospital is a major risk for the weakened patients, also for the certain places where are practiced the care or invasive acts. Their gravity a real problem of Public health. In first rowof morbidity, mortality. The causes are often multiple: air, water, renovation work without taking precautions standards, the cases of fungal contamination and the cases of fungal contamination we declared follow in serology realized.

Materials and methods In our series from september 2011 to Mars 2013, a study was undertaken to investigate the environmental fungal flora, and systhematique serology in some units of CHU The samples sent to Laboratory for Parasitology and Mycology mycological analysis. A questionnaire was conducted in which there is information for each sample .patients answering war the usual protocols of treatments Antibiotics and a fever it was asked to realize takings in aimed fungal at these patients: blood test on tube dry,. The takings treated in direct examination and in culture on specific Common agreement with the doctors (Heads of the services an environmental study is realized (harnessing of the environmental fungal flora in the respective services as well as a follow-up of the serology of the patients.Galactomannane and Antibody

Results The patients in question benefited from a serology Aspergillosis Elisa and from an antifungal treatment with one followed by the serology.

Conclusion The fungal infection is always relegated to the last rows, by misunderstanding or by absence of service competent in clinics. Nevertheless must be considered in front of a therapeutic failure in antibiotics in such departments at risks.

Pharmacogenetics of micro RNAs in Osteosarcoma

Bilbao-Aldaiturriaga Nerea,Lopez-Lopez Elixabet, Martin-Guerrero Idoia, Gutierrez-Camino Angela, García-Orad Africa, Zalacaín-Díez Marta Patiño-García Ana

Purpose Methotrexate (MTX) is a key component in the treatment against osteosarcoma. Treatment with high-dose MTX (HD-MTX) often causes toxicity. In those cases, dose reduction or cessation of treatment is needed. In the last years, the relationship between genetic variation and MTX-toxicity has been investigated, but most of these studies are focused in coding regions. Recent studies have provided evidence that SNPs in pre-miRNAs and in genes of miRNA processing machinery may affect the expression of genes involved in drug response. In fact, MTX transporters as ABCC2 and ABCC4 could be regulated by miRNAs. The aim of this study was to determine the potential role of SNPs in pre-miRNAs or their processing genes as markers of MTX-toxicity in children with osteosarcoma.

Materials and Methods We analyzed 99 blood samples from osteosarcoma patients. MTX plasma levels were used as objective and quantifiable toxicity markers. Cut-off values for toxicity were set at 3,5x10-6 at 24h and/or 3,5x10-7 at 48h post-infusion. We studied 118 SNPs in premiRNAs and in genes of the miRNA biogenesis pathway with the Taqman OpenArray platform.

Results Of the 118 SNPs analyzed, 9 were significantly associated (p<0,05) with MTX plasma levels. One of them was located in a pre-miRNA (mir-423) and eight in miRNA biogenesis pathway genes (EIF2C1, DROSHA, CNOT4, CNOT1, SND1 and XPO5).

Conclusion Our results suggest that polymorphisms in both pre-miRNAs and processing machinery genes may affect toxicity due to HD-MTX in osteosarcoma.

This project was supported by RETICS (RD/06/0020/0048), UPV/EHU (UFI 11/35) and Basque Government (IT661-13). Support by SGIker (UPV/EHU) is gratefully acknowledged.

Association of CYP2C19*2 variant with low response to clopidogrel

Botton Mariana Rodrigues, Bandinelli Eliane, Manica André Luiz Langer, Polanczyk Carisi Anne, Sarmento-Leite Rogério, Hutz Mara Helena

Clopidogrel is used in the treatment of arterial thrombosis related disorders, such as acute myocardial infarction and prophylaxis after angioplasty. However, clopidogrel therapy is associated with 10% of recurrent ischemic events and patients with non-responsive phenotype have a great risk to develop a negative outcome. The prevalence of this phenotype is about 15-30%. As a result, a substantial proportion of patients do not have benefits from the treatment. CYP2C19 enzyme is responsible for the transformation of clopidogrel to its active metabolite. The CYP2C19*2 genetic variant is associated with reduced enzymatic activity. The aim of the present study was to verify if this polymorphism is associated with clopidogrel non-responsive phenotype. We studied 94 patients that were using clopidogrel for at least seven days. VerifyNow was used to analyze the platelet aggregation of patients. Genotypes of CYP2C19 were determined by allelic discrimination with TaqMan assays. The non-responsive phenotype was found in 35% of patients, considering as a cut-off PRU=230. The average PRU was 205. Most patients were male (63%) and euro-descendants (87%). CYP2C19*1/*2 and CYP2C19*2/*2 genotypes were more frequent in the non-responsive group compared with the responsive one (31,3% and 9,4% vs. 22,6% and 0%, respectively; P=0,025). In addition, carriers of CYP2C19*2 variant had higher average PRU than patients with CYP2C19*1/*1 genotype (228 vs. 191, respectively; P=0,039). Therefore, we suggest that CYP2C19*2 variant has an important role in clopidogrel effect.

Impact of the MTHFR, MDR1 and GGH polymorphisms on response to methotrexate treatment in RA Algerian population

Boughrara Wefa, Fodil Mostefa, Aberkane Meriem, Benzaoui Ahmed, Zemani Faouzia, Dahmani Chahinez, Petit teixeira Elisabeth, Abdallah Boudjema,

Methotrexate (MTX) is the first line treatment for rheumatoid arthritis. Many aspects about the pharmacology of MTX are not clear. As an analog of dihydrofolic acid, it generates an anti-inflammatory effect, catalyzes the conversion of homocysteine to methionine and prevents the conversion of UMP to TMP. Numerous studies have shown polymorphisms genes regulating enzymes in the méthotrexate metabolic pathways. Many of these polymorphisms are related to the effectiveness and safety of drugs, but the available proof is not conclusive yet. The aim of this study is to determine the impact of C677T (rs1801133) and A1298C (rs1801131) polymorphisms in the methylenetetrahydrofolate reductase gene (MTHFR), C452T (rs11545078) polymorphism in gamma glutamyl hydrolase (GGH) and C3435T (rs1045642) polymorphism in multidrug resistance 1 (MDR1) on efficacy/toxicity of MTX. One hundred teen (n=110) patients with RA and one hundred (n=100) controls are enrolled in the study. The efficacy is evaluated using the disease activity score 28 according with EULAR criteria. Patients are genotyped for C677T, A1298C and C3435T polymorphisms by real time PCR (Taqman), and for C452T by PCR Risa/RFLP. Based on EULAR criteria, 61.38% RA patients are responder and 38.61% RA patients are non-responder of MTX therapy. Our findings suggest that there is no evident distribution of alleles/genotypes of these polymorphisms between responder/non responder and presence of ADEs/absence of ADEs in RA patients with MTX treatment. This study revealed, for the first time in the Algerian population that none of the four polymorphisms tested show any association with efficacy and toxicity of MTX treatment in RA patients.

Implications of IL28-B-gene single nucleotide polymorphisms in pharmacologic treatment of HCV+ patients: An Italian study

Carboni Ilaria, Giuliani Costanza, Meli Massimo, Bartalesi Filippo, Corti Giampaolo, Torricelli Francesca

Hepatitis C virus (HCV) infects more than 175 million people worldwide, being one of the most common transmitted diseases. In the last years, single nucleotide polymorphisms (SNPs) upstream IL28-B gene have been related to treatment-induced clearance of HCV infection caused by genotype 1 HCV. The rs12979860 and rs8099917 SNPs are implicated in long term sustained virological response (SRV) rate after treatment with pegilated interferon-alpha (PEG-IFN) and ribavirin . A population of 100 HCV+ Caucasian subjects has been screened for rs12979860 and rs8099917 SNPs. SNPs detection has been performed by means of a Real-Time PCR commercial kit. Only 19.8% of patients shows the IL28-B wild-type genotype (rs12979860CC/rs8099917TT), while the 1.2% carries both SNPs in homozygous status (rs12979860TT/rs8099917GG genotype). A total of 24.7% has a heterozygous genotype, showing one IL28-B SNP (18.6% rs12979860CT/rs8099917TT genotype, 6.1% rs12979860TT/rs8099917TG genotype). The most interesting data concerns 54.3% of individuals carrying both SNPs in heterozygous status (rs12979860CT/rs8099917TG genotype); this percentage is higher than expected in Caucasian population. The aim of the study was evaluating in a retrospective way patient’s treatment response in correlation with IL28-B genotype and clinical disease stage.

Pharmacogenetics of CYP2C8 influences selection of malaria parasite resistance associated alleles

Cavaco Isa, Mårtensson Andreas, Fröberg Gabrielle, Msellem Mwinyi, Björkman Anders, Gil José Pedro

Malaria is one of the most lethal infectious diseases in the world being responsible for more than half million deaths every year. Despite all the efforts of developing a vaccine this is still not a reality and chemotherapy represents the major strategy for malaria treatment and control. Artemisinin combination therapy strategies are based on the fast acting artemisinin derivatives, associated to longer half-life quinolinic antimalarials have been successfully implemented as global strategy for malaria control. Amodiaquine (AQ) is one of the drugs used in the combination therapy being effective against chloroquine and sulfadoxine-pyrimethamine resistance parasites. AQ is rmetabolized to a main active metabolite, N-desethylamodiaquine (DEAQ) and the reaction in the liver is mainly catalyzed by the polymorphic CYP2C8. The in vivo parasite response to AQ therapy has been documented to be dependent on polymorphisms in pfmdr1 (multidrug resistance gene 1), in particular concerning the N86Y (2), Y184F and D1246Y (3,4) SNPs. We hypothesized that the CYP2C8 status of the human host may influence the post-treatment selection of these pfmdr1 polymorphisms. We have retrospectively analyzed the CYP2C8 status of 116 patients (<5 year old) from Zanzibar islands experiencing recurrent parasitemias. The CYP2C8*2 and 2C8*3 alleles were characterized applying published methods (1). These pharmacogenetics results were merged with published parasite genotype data on the pfmdr1 N86Y, Y184F and D1246Y SNPs (2, 4).

The results shown that carriers of the very low activity CYP2C8*3 had an increased frequency of parasites carring the pfmdr1 86Y (100% vs 74.6%, p<0.05) and 1246Y (46.7% vs 13.6%, p<0.01) alleles, compared with the reference homozygous wild type group (CYP2C8*1). Morover, the analysis of the 86Y/184Y/1246Y haplotype associated with in vivo selection after AQ and ASAQ exposure (2,4) shown that carriers of YYY carrying parasites had a tendency to be more frequent among the 2C8*3 carriers, when compared with the wild type group (53.3% vs 24.6%, p=0.056). In contrast with the CYP2C8*3 carrier subset, the CYP2C8*2/*2 homozygous, as well as the overall subset of 2C8*2 carriers were not associated with any of the pfmdr1 SNPs analyzed. These results support a potential influence of the host pharmacogenetic on the long-term selection of parasite characteristics associated with antimalarial drug response.

  1. Cavaco I, Strömberg-Nörklit J, Kaneko A, Msellem MI, Dahoma M, Ribeiro VL, Björkman A, Gil JP. Eur J Clin Pharmacol. 2005; 61:15-8.

  2. Holmgren, G., J. P. Gil, P. M. Ferreira, M. I. Veiga, C. O. Obonyo, and A. Björkman. 2006. Infect. Genet. Evol. 2006; 6:309–314.

  3. Humphreys GS, Merinopoulos I, Ahmed J, Whitty CJ, Mutabingwa TK, Sutherland CJ, Hallett RL. Antimicrob Agents Chemother. 2007; 51:991-7.

  4. Holmgren G, Hamrin J, Svärd J, Mårtensson A, Gil JP, Björkman A. Infect Genet Evol. 2007; 7:562-9.

Customized first line chemotherapy according to excision repair cross complementation group 1 (ERCC1) and ribonucleotide reductase M1 (RRM1) Single Nucleotide Polymorphisms (SNPs) in advanced Non-Small-Cell Lung Cancer (NSCLC) patients: A phase II study

Giuliani C, Mazzoni F, Carboni I, Cecere FL, Meoni G, Boni L, Camerini A, Allegrini G, Amoroso D, Martella F, Giommoni E, Di Costanzo F, Torricelli F

ERCC1 and RRM1 gene expression is predictive of chemotherapy (CT) efficacy in NSCLC. Through a phase II trial, a first-line CT was planned in pts with NSCLC according to ERCC1 (118T/C, 8092C/A) and RRM1 (-37C/A, -524T/C) SNPs related to specific expression levels. CT delivered was: treatment A (low ERCC1 and RRM1) Cisplatin+Gemcitabine; treatment B (low ERCC1, high RRM1) Cisplatin+Docetaxel; treatment C (high ERCC1, low RRM1) Gemcitabine+Docetaxel; treatment D (high ERCC1 and RRM1) Docetaxel+Vinorelbine. 40 pts received at least 1 cycle of CT; median age was 66 years (range 47-72); 30 (75%) pts were males; 21 (52%) pts showed ECOGPS0, 19 (48%) PS1; 36 (90%) pts had stage IV, 4(10%) IIIB; 23(58%) pts had adenocarcinoma, 14 (35%) squamous; 25 (62%) pts received treatment A, 3 (8%) treatment B, 11 (27%) treatment C, 1 (3%) treatment D. Here we report the first step analysis results. The overall best response has been assessed as primary end-point; we found a 55% response rate (RR) [38.7–70.4;95%CI] in the intention-to-treat population. Subgroups analysis showed 71.4% RR in squamous pts. Secondary end-points were progression free survival (PFS), overall survival (OS) and safety. The median follow-up time is 10 months, PFS is 5.3 months (95% CI: 3.5-6.0) and OS is 8.0 months (95% CI: 7.3-12.6). CT was well tolerated: 17(42%) pts showed grade 3-4 toxicity and there were no treatment related deaths. We observed an increase of RR in NSCLC pts treated with CT according to ERCC1 and RRM1 SNPs status.

Influence of IL-6 -174 G>C Polymorphism in the overall survival of Non-Small Cell Lung Cancer patients

Gomes Mónica, Coelho Ana, Araújo António, Teixeira Ana L, Catarino Raquel, Medeiros Rui,

Introduction Lung cancer is the leading cause of death by cancer in the world, originating about 17.5% of total deaths from cancer (1.18 million). Inflammation and related pathways play an important role in the pathogenesis of lung cancer. IL-6 is a potent pleiotropic inflammatory cytokine that is considered a key growth-promoting and antiapoptotic factor. IL-6 is of particular interest because they are expressed in malignant epithelial cells, and their expression is associated with a poor prognosis in lung cancer patients. The - 174G>C polymorphism (rs 1800795) of the IL6 gene controls serum levels of IL6 and may be associated with cancer risk, but the results from the published studies on the association between this polymorphism and cancer risk are conflicting. The G allele increases IL-6 expression, both in basal and stimulated conditions, the highest IL-6 levels in plasma and serum being found in subjects homozygous for the G allele. The aim of this study was to evaluate the influence of this polymorphism in the overall survival of non-small cell lung cancer (NSCLC) patients.

Material and Methods Caucasian patients (n=424) admitted to the Portuguese Institute of Oncology of Porto (IPO-Porto), Portugal, with cytological or histological confirmed NSCLC, have been prospectively recruited to the study (1997-2010) (mean age 62.5 years; sd 10.3). DNA was extracted from peripheral-blood samples of individuals. The characterization IL-6 - 174G> genotypes was performed by PCR-RFLP (NlaIII). Overall survival (OS) was the endpoint of this analysis and was calculated from the date of diagnosis to date of death of the patient. Data were collected from patients medical records. The associations between IL-6 polymorphisms and overall survival were estimated by Kaplan-Meier methodology and assessed using Breslow Test. Survival data were analyzed according to IL-6 polymorphisms’ genotypes.

Results The IL-6 - 174G>C polymorphism was significantly associated with overall survival in the NSCLC patients. IL-6 polymorphism’s genotypes was divided according functional activity, so G carriers (CG/GG) and CC genotype. According to this split, patients with genotypes carrying the G allele (CG/GG) had a statistically significant diminished survival when compared with patients with CC genotype (42.3 months and 62.7, respectively; P=0.042).

Conclusion The results obtained in this study suggest an association between IL-6 polymorphisms and overall survival in patients with NSCLC. However, the IL-6 status was accessed in blood samples, so further studies should be conducted in tumour tissue samples, to confirm these results. Moreover, controversial results have been published in literature regarding the influence of the studied variants in IL-6 expression in the tissue and its serum levels. In the era of pharmacogenomic profiles and directed therapies, it would be important to conduct further functional studies are to clarify the role of this polymorphism in IL-6 expression and how it conditions tumor progression, in order to better understand the observed effect.

The influence of polymorphisms in folate pathway genes on pemetrexed treatment outcome in malignant mesothelioma patients

Goričar Katja, Kovač Viljem, Dolžan Vita

Malignant mesothelioma (MM) is a rare but aggressive tumor of mesothelial surfaces of pleura associated with asbestos exposure. A randomized study has shown that treatment with combination of pemetrexed (PMX) and cisplatin improves outcome in MM patients. PMX is a folic acid analogue that inhibits several key folate pathway enzymes, leading to impaired DNA synthesis. Genetic variability in folate pathway genes may contribute to interindividual differences in response to PMX between patients. The aim of our study was to evaluate the influence of polymorphisms in folate pathway genes on treatment outcome in Slovenian patients with MM.

In total, 41 patients with MM receiving PMX were genotyped for six polymorphisms in five folate pathway genes: MTHFR rs1801133 and rs1801131, TYMS rs34743033, MTHFD1 rs2236225, MTR rs1805087, and MTRR rs1801394. Logistic regression was used to assess the influence of polymorphisms on treatment efficacy and toxicity, while Cox regression was used to determine their influence on progression-free and overall survival.

None of the investigated polymorphisms was associated with the occurrence of PMX-related toxicities, but MTHFD1 rs2236225 affected both treatment response and survival of MM patients. Patients with at least one polymorphic MTHFD1 rs2236225 allele had a significantly lower response rate (P=0.005; OR=0.12; 95% CI=0.03-0.54) and shorter progression-free survival (P=0.032; HR=3.10; 95% CI=1.10-8.74; adjusted for clinical variables) than patients with two wild-type alleles.

Our results indicate MTHFD1 rs2236225 might serve as a potential marker for the prediction of PMX treatment outcome in MM patients and thus contribute to more personalized treatment of MM.

Single Nucleotide Polymorphism of UGT1A4 enzyme in a Turkish population of patients with epilepsy on lamotrigine therapy

Gulcebi Medine I, Ozkaynakci Aydan, Goren M Zafer, Ozkara Cigdem, Onat Filiz

Genetic polymorphism of the metabolism enzymes can lead to adverse reactions or inefficiency of the drugs by affecting the serum concentration levels. An antiepileptic drug with a widespread use, lamotrigine (LTG), is metabolized by glucuronidation conjugation with primarily UDP-glucuronosyl transferase (UGT) 1A4 enzyme. LTG has no effect on the metabolism of other antiepileptic drugs however the serum concentration of LTG has been shown to increase with valproic acid (VPA) or decrease with hepatic enzyme-inducing drugs such as phenytoin, carbamazepine and phenobarbital. The most frequent adverse effect of LTG is rash which can lead to very serious syndromes such as Steven-Johnsons or Lyell syndromes. This study aimed to investigate the effect of single nucleotide polymorphism, P24T, of the UGT1A4 enzyme (UGT1A4*2), in a Turkish population of epilepsy patients on monotherapy (n=35) or polytherapy with LTG (n=94). Patients needed to be on LTG treatment for at least two weeks so that they were in a steady state of drug levels. Patients who did not want to participate in the study, patients with chronic hepatic or renal disease, with an improper use of LTG were excluded from the study. Venous blood samples were collected immediately before the last LTG dose. Serum levels of LTG were measured with high performance liquid chromatography and the analysis of the P24T polymorphism was made with a matrix assisted laser desorption-time of flight mass spectrometry method. According to the genotype results, 3.8 % of patients were found to be heterozygous for the P24T polymorphism. Of the 5 polymorphic patients 3 were on polytherapy with LTG and VPA whereas 2 were on polytherapy with LTG, VPA and hepatic enzyme-inducing drugs. Comparison of the serum LTG (1-3 mg/kg/day) levels of the VPA add on therapy patients between wild type (4.8 mg/L) and polymorphic alleles (3.8 mg/L) with one-way ANOVA and post-hoc Bonferroni test were found not to be significant. Serum level of LTG of the polymorphic patient on hepatic enzyme-inducing antiepileptics add on therapy was 3.1 mg/L whereas for the other polymorphic patient using LTG, VPA and hepatic enzyme-inducing drugs, serum LTG level was 11.2 mg/L. As a result although the increasing effect of VPA on the serum LTG level was shown to have tendency to decline in the UGT1A4*2 alleles, it is difficult to determine the exact effect of this polymorphism on serum LTG level with this low frequency.

Pharmacogenomics activity of thyme extract in rats fed aflatoxin-contaminated diet

Hamzawy Mohamed A., Abdel-Azim Sekena H., Hassan Aziza M., El-Denshary Ezzeldein S., Abdel-Wahhab Mosaad A.,

The aims of the current study were to evaluate the possible pharmacogenomics activity of thyme leave extracts on AFs contaminated diet that may lead to oxidative stress, genotoxicity and alteration of p53 bax and bcl2 gene expressions. Fourty male Sprague-Dawley rats were divided into four equal groups included: the control group, the group fed AFs-contaminated diet (2.5 mg/kg diet) for five weeks, the groups treated orally with thyme extract (0.5 g/kg b.w) for six weeks and the groups pretreated orally with thyme extract one week before and during AFs treatment for other five weeks. Blood, liver and bone marrow samples were collected for biochemical analysis, gene expression, DNA fragmentation and micronucleus assay. The results showed that AFs induced significant alterations in oxidative stress markers, increased serum AFP and inflammatory cytokine, percentage DNA fragmentation, the expression of pro apoptotic gene p53 and bax accompanied with a decrease the expression of bcl2. Animals treated with the extracts one week before AFs treatment showed a significant decrease in oxidative damage markers, micronucleated cells, DNA fragmentation and modulate the expression of pro apoptotic genes. These results suggested that thyme extracts had pharmacogenomics activity and modulatory effect through alteration of genetic toxicity due to their higher content of total phenolic compounds.

Pharmacogenomics translation; From bench to bedside

Hamzawy Mohamed A.

Pharmacogenomics is a new approach that elucidates the role of genetic materials of how genetic variation relates to the drug efficacy and safety. The premium goal of pharmacogenetics is a personalized treatment, in where according to genotype; it could be able to prescribe the most effective drug at the most appropriate dose for an individual patient, improve the ability to predict drug response with proper therapeutic drug monitoring. Over the past decade, pharmacogenetics incorporated into pharmacologic research and drug development initiatives in hope of improving medical care or economical benefits. The issue in drug development is not only the identification of genetic biomarkers for drug efficacy and safety, but it also includes development and distribution of the methodology for reliable detection of these biomarkers in laboratories in real clinical settings, and also proper education of clinicians assuring the adherence to the pharmacogenetic screening. Authorization agencies world-wide recognized the importance of pharmacogenomic during drug development. It is now evident that number of medicines that were granted marketing authorization status would not be licensed if there were no pharmacogenetic biomarkers for the prediction of efficacious and safe use. Pharmacogenetics is a discipline that should be validated from cell to clinical practice, genetic testing methods based on major cellular component; DNA. The polymerase chain reaction (PCR), a procedure that exponentially amplifies a piece of DNA of specific size and sequence is characterized by especially rapidity, specificity, sensitivity, sample accessibility, low cost and simplicity. PCR can be used in different methods either simple such as allele-specific PCR or more complex such as real-time PCR. Another method has been introduced to detect single nucleotide polymorphism (SNP), which can be analyzed with rapid using and minimum of biological material and of course time saving. In this concern, based on the growing evidence patients can be divided into four classes according to their single locus genotype: “poor” or “slow metabolizers” (PM), “intermediate metabolizers” (IM), “extensive metabolizers” (EM) and “ultra rapid metabolizers” (UM). Fast metabolizers are the most frequent in the population and they are considered as a standard group. However, genetic testing should be performed for three reasons; (1) selection of patients with the highest probability of therapeutic efficacy, (2) reduction of adverse drug reactions (ADRs) and (3) determination of the most appropriate drug dosage to provide efficacy and safety of the treatment. Recent studies has been suggested that most prescribed medications are effective in no more than 60% of the individuals in whom they are used and a significant number of patients develop major adverse effects often leading to hospitalization. Indeed, real stories have been published about pharmacogenomics such as warfarin therapeutic failure; a dosage story with widely variable prescribed doses. Although pharmacogenetics is already starting to influence how physicians and scientists design clinical trials and despite its impact on the practice of medicine, the task of developing individualized therapy tailored to patient’s genotypes poses still a major scientific challenge and offers many opportunities for research.

The influence of genetic variability in methotrexate transporters on toxicity in rheumatoid arthritis

Jenko Barbara, Lusa Lara, Tomsic Matija, Praprotnik Sonja, Logar Dusan, Dolzan Vita

Introduction Methotrexate (MTX) is one of the most commonly used disease-modifying anti-rheumatic agents. Generally it is well tolerated, although a number of potentially serious adverse effects and/or inefficacy of treatment can occur. It is becoming widely accepted that not a single gene but several gens coding for MTX metabolizing enzymes, transporters, targets and signaling pathways in combination with clinical factors may determine MTX response in rheumatoid arthritis (RA) patients. Aim: To evaluate the influence of polymorphisms in genes coding for MTX transporters on MTX discontinuation due to toxicity and on occurrence of specific toxicity in Slovenian patients with RA. Methods: The study included 212 RA patients treated with low dose MTX. The data on sex, age, disease and treatment duration at inclusion, MTX dose, presence of bone erosions, RF or anti-CCP seropositvity and mean DAS28 were obtained from the medical records. ABCB1 rs1128503, ABCC2 rs2273697, rs2804402, rs717620, ABCC4 rs2274407, ABCG2 rs2231137, rs22311342 and SLCO1B1 rs4149056, rs11045879, rs2900478, rs2306283 polymorphisms were genotyped using allele-specific real time polymerase chain reaction (KASPar Assay). The combined effects of genetic and clinical factors were analyzed with lasso penalized regression.

Results When all clinical and genetic factors were entered in the penalized model only mean DAS28 (HR: 1.73) and MTX dose (HR: 1.09) significantly influenced the risk on MTX discontinuation due to toxicity. When we analyzed with linear regression models higher risk for hepatotoxicity was associated with SLCO1B1 rs2900478 (p = 0,036). When all genetic and clinical factors were included in model, the penalized regression analysis showed the influence of SLCO1B1 rs2900478 (OR: 1.41), ABCB1 rs1045642 (OR: 1.29), ABCC2 rs717620 (OR: 1.23) and mean DAS28 (OR: 1.29) on hepatotoxicity. Bone marrow toxicity was significantly higher in carriers of polymorphic ABCC2 rs2804402 allele than in non-carriers (p=0.050). In penalized regression model ABCC2 rs2804402 (OR: 2.04), ABCB1 rs2032582 (OR: 1.24), SLCO1B1 rs4149056 (OR: 0.88) and mean DAS28 (OR: 0.94) were associated with bone marrow toxicity.

Conclusion Genetic variability in MTX transporters significantly influenced MTX toxicity in RA patients. Our results support the use of lasso penalized regression in analyses of combined effect of genetic and clinical factors on treatment outcome.

From pharmacogenetics to pharmacometabolomics: TPMT and SAM

Karas Kuzelicki Natasa, Smid Alenka, Tamm Riin, Metspalu Andres, Mlinaric Rascan Irena

Dosage individualization of 6-MP therapy in childhood ALL is largely dependent on thiopurine S-methyl-transferase (TPMT) phenotype. Determination of mutations in TPMT gene before starting 6-MP therapy could represent a quick, simple and cost-effective strategy to individualize thiopurine dosing and thus lower the incidence of side effects and increase the efficacy of the treatment. However, in a certain number of patients, prediction of TPMT enzyme activity and response to therapy are not possible purely on the basis of genotype. Therefore, it is of great importance to search for additional factors, which might influence TPMT activity (1, 2). In 2005 Arenas M et al. suggested for the first time that the TPMT cofactor S-adenosylmethionine (SAM) and enzyme involved in its biosynthesis, methylene-tetrahydrofilate reductase (MTHFR), could influence TPMT activity (3). The binding of SAM had been shown previously to stabilise the 3D structure of the enzyme (4). We confirmed these findings on cell lines (5, 6) and in a retrospective study on ALL patients (2). However, the direct influence of SAM on TPMT activity in vivo on human subjects has not yet been studied and confirmed. In cooperation with Estonian genome project of University of Tartu 1017 blood samples were collected from healthy volunteers, donors of the Estonian Biobank. All samples were genotyped for common TPMT variants using TaqMan probes. TPMT activity and SAM levels were measured in RBCs hemolysates using HPLC method. A set of 13 biochemical and 10 hematological parameters were determined in plasma and blood of donors, respectively. The demographic data for donors (including age, gender, smoking and eating habits, etc) was extracted from the Biobank database. After adjustment in multiple regression model and correction for multiple testing, from 43 factors tested, only TPMT genotype (p = 1×10-13) and SAM levels (p = 1×10-13) were found to significantly influence TPMT activity. Furthermore, the subjects with normal TPMT activity despite mutated TPMT exhibited very high correlation between the TPMT activity and SAM levels (r = 0,879, p = 0,00081), indicating that discrepancy between TPMT genotype and activity in TPMT mutated individuals might be due to different SAM levels. Therefore, from the diagnostic and economic point of view, it is reasonable to measure SAM levels only in TPMT mutated patients. TPMT mutated patients with high SAM levels could be given standard doses of 6-MP to improve the efficacy of the therapy, while in TPMT mutated patients with low SAM levels, SAM supplementation during 6-MP therapy could lower the incidence of side effects.

1. Karas Kuzelicki et al. Pharmacogenomics 2009; 10(8): 1309-1322

2. Karas Kuzelicki et al. Leukemia 2009; 23: 971-974

3. Arenas M et al. Clin Chem 2005; 51(12): 2371-2373

4. Scheuermann et al. Biochemistry 2004; 43: 12198-12209

5. Milek et al. Biochem Pharmacol 2009; 77: 1845-1853

6. Milek and Smid et al. Biochem Pharmacol 2012; 83: 969-976

Validation of a TaqMan Real-Time PCR method for the genotyping of the inosine thriphosphate and solute carrier genes

Kheloufi Farid, Quaranta Sylvie, Lacarelle Bruno, Solas Caroline

Inosine triphosphatase (ITPA) is an enzyme that catalyzes the conversion of inosine triphosphate (ITP) and deoxy-inosine triphosphate (dITP) to inosine monophosphate and deoxy-inosine monophosphate, respectively, thereby maintaining low intracellular concentrations of ITP and dITP. It has been shown that a congenital deficiency in ITPA could lead to an accumulation of ITP inside red blood cells and thus be protective against ribavirin induced-anemia in HCV infected patients. In addition, others polymorphisms of solute carrier genes, SCL29A1 and SCL28A3, coding for RBV transporters, respectively ENT (equilibrative nucleoside transporter) et CNT (concentrative nucleoside transporter) would be associated to either rapid virological response or anemia in HCV infected patients treated by pegylated-interferon and ribavirin. Our aim was to validate a genotyping method of ITPA and SLC genes in order to apply it to HIV-HCV coinfected patient’s blood samples treated by ribavirin. We focused on two variants of the ITPA gene (rs1127354 and rs7270101) and three variants of the SLC gene (rs10868138 and rs56350726 for SLC28A3, rs760370 for SLC29A1), which were the most significant in term of protecting effect towards ribavirin induced-anemia and/or rapid virological response to treatment. We decided to use a Taqman real-time PCR assay on LightCycler® 480 (Roche Diagnostics Instruments) because of the availability of kits designed especially for these single nucleotide polymorphisms (SNP). Human genomic DNA was extracted with an automatic extraction system from blood samples previously used to validate another genotyping assay. Our method was validated according to the EMA Guidelines on bioanalytical method validation. Each amplification reaction was carried out in a total volume of 10 µL, in presence of 5 µL of genotyping master mix (2X), 0.25 µL or 0.5 µL of SNP genotyping assays (40X or 20X), 3.75 µL or 3.5 µL of water for PCR and 1 µL of the DNA sample. Concentrations of reagents were optimized for each SNP. Among 13 samples analyzed for ITPA, only one was heterozygous for the both variants, five samples were heterozygous for the rs7270101 variant and four samples were heterozygous for the rs1127354 variant. Among four samples analyzed for SLC, all samples were heterozygous for the rs10868138 variant and wild-type for the rs56350726 variant. One homozygote mutated, one heterozygous and two wild-type profiles were identified for the rs760370 variant. A cross-validation with a laboratory using the reference method for genotyping (sequencing) was realized for the ITPA variants and we obtained 100% of matching results. The method was found robust for the two zones of fluorescence [465-510] and [533-580]. Therefore, the method was successfully validated and we’re currently waiting for blood samples from two studies: a local retrospective study in patients treated with ribavirin/pegylated-interferon and the clinical ANRS-HC27 BOCEPREVIH trial (patients treated with ribavirin/pegylated-interferon/boceprevir) to perform these genotyping in order to assess the relationship between these variants, ribavirin concentration and ribavirin-induced anemia and/or virologic response.

Roles of folate pathway gene variations in susceptibility to childhood acute lymphoid leukemia

Lautner-Csorba Orsolya, Gézsi András, F.Semsei Ágnes, Kutszegi Nóra Z., Erdélyi Dániel, Kovács Gábor T.

Background Acute lymphoid leukemia (ALL)is the most common haematological maignancy in childhood. The aim of our study was to investigate the influence of single nucleotide polymorphisms (SNP) in genes of folate cycle and transporter molecules on the pharmacogenomics of childhood acute lymphoblastic leukemia. Folate metabolism has a critical role in de novo DNA synthesis and methylation process. Genetic variations of key regulators of this pathway, such as methionine synthase reductase (MTRR) and methylene tetrahydrofolate reductase (MTHFD1) may influence the susceptibility to pediatric ALL. Methods: We carried out a retrospective study included 543 ALL patients and 529 controls. 64 genetic polymorphisms in 15 candidate genes were selected and investigated whether the presence of these polymorphisms was associated with the disease. DNA samples were isolated from peripheral blood collected in ten Hungarian haematology centers. Genotyping was performed with Sequenom iPLEX Gold MassARRAY technology (McGill University and Génome Québec Innovation Centre, Canada). For statistical evaluation we applied frequentist and Bayesian network-based Bayesian multilevel analysis of relevance (BN-BMLA) methods.

Results We demonstrated the significant effect of two polymorphisms, the rs1076991 in MTHFD1 and rs3776455 in MTRR genes related to ALL susceptibility. We found that patient carrying the rs1076991 GG genotype (p=3.52E-04; OR=2.00) might have a significant risk for developing B-ALL. However in case of children with the variant rs3776455 GG genotype (p=1.21E-03; OR=0.55) in MTRR gene had lower risk to the disease. We also analysed the survival rate of ALL patients and found a slightly significant association with a lower overall survival rate of rs9909104 TC genotype compared to TT genotype (80.2% vs. 88.8%; p=0.01) in SHMT1 gene. After the BN-BMLA evaluation we gained consistent resuls with the frequentist technique. By analysing the results with BN-BMLA we tried to estimate whether a variable was directly relevant or its association was only mediated. MTHFD1 and MTRR gene polymorphisms seemed to be strongly relevant to ALL. Besides these results, polymorphisms in DHFR, GGH, SLCO1B1, and TYMS showed association with acute lymphoid leukemia.

Conclusion As a useful supplementary tool of frequentist statistics, BN-BMLA offers an automated and rich language for the detailed representation of types of relevance, including direct and indirect aspects. Results of this study might provide a rationale for evaluating such variants as prognostic factors.

Reduced activity of cytochrome P450 2C9*35 allelic variant is determined by the altered interaction with P450 oxidoreductase

Lee Mi-Young, Borgiani Paola, Johansson Inger, Mkrtchian Souren, Falconi Mattia, Ingelman-Sundberg Magnus

Cytochrome P450s (CYP) are hepatic monooxygenases that metabolize endogenous compounds and drugs. Cytochrome P450 2C9 (CYP2C9) is one of the abundant CYPs, which hydroxylates many clinically important drugs including warfarin, losartan and diclofenac. We have recently reported a new allelic variant, CYP2C9*35, characterized by the Arg125Leu and Arg144Cys amino acid changes found in the warfarin hyper-sensitive Italian patient (1). It was suggested that such phenotype might be connected with the reduced activity of CYP2C9, a hypothesis that we have tested in current work. CYP2C9.1, CYP2C9-R125L, CYP2C9-R144C (CYP2C9.2), CYP2C9.35 constructs were transfected into HEK293 cells and the activity of the expressed enzymes was analyzed in the isolated microsomes incubated with diclofenac and NADPH as an electron donor. CYP2C9.1 and CYP2C9.2 proteins were found catalytically active. However, diclofenac was not metabolized by CYP2C9-R125L or CYP2C9.35. In order to understand whether this is connected with the altered substrate-CYP interaction or failure of CYP to recognize its redox partner, P450 oxidoreductase (POR), we used cumene hydroperoxide as electrone donor to the enzyme, bypassing thus the POR. Indeed, in such experimental setup all variant CYP2C9 enzymes effectively oxidized diclofenac suggesting that the Arg125Leu mutation does not affect the catalytic activity of the enzyme per se, but rather alters CYP-POR interaction. This was confirmed by in silico analysis showing decrease of electrostatic potential of the proximal surface of CYP2C9-R125L that interacts with POR with the ensuing disruption of the important salt bridges between the proteins, whereas the substrate binding loci remained intact.

  1. Ciccacci C, Falconi M, Paolillo N, Oteri F, Forte V, Novelli G, Desideri A and Borgiani P, Characterization of a novel CYP2C9 gene mutation and structural bioinformatics protein analysis in a warfarin hyper-sensitive patient, Pharmacogenet Genomics. 2011; 21: 344-346

MicroRNA-related SNPs and toxicity in pediatric Acute Lymphoblastic Leukemia

Lopez-Lopez Elixabet, Gutierrez-Camino Ángela, Martín-Guerrero Idoia, Piñán M Ángeles, Uriz Javier, Navajas Aurora, García-Miguel Purificación, Ballestero Javier, García-Orad África

Despite the clinical success of acute lymphoblastic leukemia (ALL) therapy, toxicity is frequent. Therefore, it would be useful to identify predictors of adverse effects. In the last years, several studies have investigated the relationship between genetic variation and treatment-related toxicity. However, most of these studies are focused in coding regions. Nowadays, it is known that regions that do not codify proteins, such as microRNAs (miRNAs), may have an important regulatory function.

MiRNAs can regulate the expression of genes affecting drug response. In fact, the expression of some of those miRNAs has been associated with drug response. Genetic variations affecting miRNAs can modify their function, which may lead to drug sensitivity. The aim of this study was to detect new toxicity markers in pediatric B-ALL, studying miRNA-related polymorphisms, which can affect miRNA levels and function.

We analyzed 118 SNPs in pre-miRNAs and miRNA processing genes in association with toxicity in 152 pediatric B-ALL patients all treated with the same protocol (LAL/SHOP).

Among the results found, we detected for the first time an association between rs639174 in DROSHA and vomits that remained statistically significant after FDR correction. DROSHA had been associated with alterations in miRNAs expression, which could affect genes involved in drug transport. This suggests that miRNA-related SNPs could be a useful tool for toxicity prediction in pediatric B-ALL.

This project was supported by RETICS (RD/06/0020/0048), UPV/EHU (UFI 11/35) and Basque Government (IT661-13, SAI10/03 and 2006111015). Support by SGIker (UPV/EHU) is gratefully acknowledged.

New genetic markers for vincristine toxicity in childhood Acute Lymphoblastic Leukemia

Lopez-Lopez Elixabet, Echebarria Aizpea, Askaiturrieta Ziortza, Pombar Maria, Bilbao Nerea, Piñán M Ángeles, García de Andoin Nagore, García-Miguel Purificación, Navajas Aurora, García-Orad África

Acute lymphoblastic leukemia (B-ALL) is the most common pediatric malignancy. Therapeutic advances have increased survival, due in part to standardized treatment protocols. Vincristine is used in different phases of treatment (induction, intensification and reinductions). However, some individuals experience neurotoxicity and there are no markers to predict it. As vincristine has a narrow therapeutic range, pharmacogenetic studies may be useful for predicting toxicity.

Only 4 studies have been performed to date, analyzing a total of 12 SNPs in 3 genes in ALL patients. These studies have produced conflicting results for CYP3A5 gene and no association with vincristine toxicity for ABCB1 and MAPT. The lack of conclusive results may be due to non-homogeneous treatment, limited samples and SNPs analyzed and/or non-accurate selection of genes. Recently, in our group, we found new methotrexate toxicity markers, analyzing in depth genes involved in methotrexate transport. So, genes involved in vincristine transport could also have a role in vincristine toxicity.

The aim of the present study was to analyze in depth the role of genetic variations in genes involved in vincristine transport as markers of neurotoxicity in a large cohort of children with B-ALL homogeneously treated. DNA was extracted from remission blood samples of 150 pediatric ALL patients, all of them homogeneously treated with LAL/SHOP protocol. We studied 138 SNPs in 4 genes involved in vincristine transport with Illumina GoldenGate platform. The association between SNPs and neurotoxicity was analyzed using the Fisher exact test.

Our results suggest that pharmacogenetic studies may be useful for neurotoxicity prediction and adjustment of vincristine treatment in pediatric ALL patients.

This project was supported by RETICS (RD/06/0020/0048), UPV/EHU (UFI11/35) and Basque Government (IT661-13, SAI10/03 and 2012111053). Support by SGIker (UPV/EHU) is gratefully acknowledged.

Retinoblastoma in Algeria

Louhibi Lotfi, Boubekeur Amina Mama, Mahmoudi Khadidja, Abderrahmanne Rym Khadidja

The retinoblastoma is a malignant intraocular tumour which generally reaches the child with a frequency from 1/15000 to 1/20000. Changes concerning the antioncogene Rb (chromosome 13q14.2), are at the origin of this cancer. The change of the two alleles of this gene is required for development of retinoblastoma. The aim of this study is research and identification of changes able to affect the gene RB at the constitutional level. Study concern 61 patients. The twenty-seven exons and promoter of Rb gene were amplified by PCR, with 15 exons studied by DGGE (Denaturing Gel Gradient Electrophoresis) and 12 by SSCP (Single Strand Conformation Polymorphism). These two techniques allow selecting cases for sequencing. The sequencing results gave nineteen different variations bases, including seven exonic changes: 5 are nonsens mutations located in 1,7,8,18 and 23 exons and two cause amino-acid substitution in exons 19 and 20 . These changes remain minority compared to the twelve intronic changes whose possible implication remains to be elucidated. Polymorphisms found in 2,3,4,11 and 17exons were already described in the literature. Neomutations detection is important because it allows on the one hand, the prevention and precocious taking care of the bearing children carrying transferred gene not having developed the disease yet and on the other hand, the screening of the asymptomatic carriers which present risk transmission of the disease to their descent.

Characterization of the functional genetic variants relevant to statin response in the Azores Islands population (Portugal)

Melo Mafalda, Balanco Letícia, Branco Cláudia C, Mota-Vieira Luisa

Statins are lipid lowering drugs considered a standard of care in many clinical settings. They are widely used for the treatment of patients with hyperlipidemia and highly effective for the primary and secondary prevention of cardiovascular disease. Simvastatin and pravastatin are the most prescribed statins. Several studies demonstrate that clinical efficacy and toxicity of statins dependent on factors, which include inter-individual variation of genes (coding for enzymes, transporters or extracellular receptors) involved on pharmacokinetics or pharmacodynamics. Since genetic variants differ between ethnic groups, here we investigate the clinically relevant pharmacogenes underlying statin drug response, in terms of efficacy (HMGCR, CETP and ApoE) and toxicity (SLCO1B1), in the Azores Islands population. The DNA of 170 blood donors was examined for eight SNPs in candidate genes: HMGCR (rs3846662, rs17238540, rs17244841), CETP (rs708272), ApoE (rs7412, rs429358), and SLCO1B1 (rs2306283, rs4149056). These SNPs were selected according to PharmGKB, Pubmed, dbSNP and SNPedia databases, and genotyped by TaqMan® Genotyping Assays (Applied Biosystems). In general, the results demonstrate that allele frequencies for the eight SNPs in Azoreans were similar to those reported for the HapMap CEU population and/or Caucasians. Regarding efficacy, HMGCR encodes the rate-controlling enzyme of the mevalonate pathway. The HMGCR haplotype, defined by TGG risk alleles, was identified by indirect inference with very low frequency (3.2%). Therefore, it allows us to expect a successful drug response. At CETP, gene coding the protein responsible for the transport of cholesterol from peripheral tissues back to the liver, the genotype B2B2 was identified with a frequency of 13.5%. This genotype is associated with higher HDL levels and lowered risk of coronary artery disease compared with B1B1, which has a frequency of 35.3%. With respect to ApoE, an extracellular receptor that transfers VLDL and chylomicrons from plasma to the liver, it can be expected that individuals with the E2 genotype (8.5%) have greater reductions in levels of LDL compared with E4 genotype (9.1%) when treated with statin therapy. In what concerns to toxicity, statin-induced myopathy and rhabdomyolysis occur in 11.0 and 3.4 per 100,000 patient-years, respectively, with a mortality rate of 10.0% for patients with rhabdomyolysis [Law and Rudnicka. 2006. Am J Cardiol, 97:S52-S60]. SLCO1B1, which encodes a protein that regulates the uptake of statins from portal blood into the hepatocyte, is considered a good myopathy risk predictor. Based on the polymorphism 521T>C known to cause high plasma levels of statins, three genotypes define the SLCO1B1 phenotype: TT (individuals with two functional alleles, 70.6%), TC (one functional allele plus one reduced-function allele, 27.6%) and CC (two reduced-function alleles, 1.8%). Thus, clinicians should be alert to statin-induced myopathy in patients with intermediate and high risk (TC and CC, respectively) and, whenever needed, proceed to dose adjustments. Our study provides an initial step in how clinicians can use a patient’s genetic makeup to provide a better and safer therapy, improving statins efficiency and reducing myopathy risk.

Bipolar disorders genetic based expression differences in peripheral blood for lithium treatment in moderate dose

Musaraj Adanela, Mulita Besa, Panariti Emi

The aim of the study is the identification of genes as early markers for lithium treatment in moderate dose in patients with bipolar disorders. Studies of families and twins have shown the importance of genetic factors affecting susceptibility to bipolar disorder and suggesting substantial genetic and phenotypic complexity. Robust and replicable genome-wide significant associations have recently been reported in genome-wide association studies at several common polymorphisms, including variants within the genes CACNA1C, ODZ4, and NCAN. Strong evidence exists for a polygenic contribution to risk (ie, many risk alleles of small effect). Although changes in peripheral blood gene-expression may not relate directly to mood symptoms, differences in treatment response at the biochemical level may underlie some of the heterogeneity in clinical response to Li. Subjects were randomized to treatment with (n=70) or without (n=45) Li. Peripheral blood gene-expression was measured before and 1month after treatment initiation, and treatment response was assessed after 6 months. In subjects treated with Li, 62 genes were differentially regulated in treatment responders and non-responders. Of these, BCL2L1 showed the greatest difference between Li responders and non-responders. These changes were specific to Li responders (n=22), and were not seen in Li non-responders or patients treated without Li, suggesting that they may have specific roles in treatment response to Lithium. A notable finding is the overlap of susceptibility between bipolar disorder and schizophrenia for several individual risk alleles and for the polygenic risk. By contrast, genomic structural variation seems to play a smaller part in bipolar disorder than it does in schizophrenia. Together, these genetic findings suggest directions for future studies to delineate the aetiology and pathogenesis of bipolar disorder, indicate the need to re-evaluate our diagnostic classifications, and might eventually pave the way for major improvements in clinical management.

Non small cell lung cancer Roma patients treated with platinum-based chemotherapy shows differences in results related to genetic polymorphism in 8 genes

Musaraj Adanela, Teodora Lentch

Aim The aim of the study is the determination of pharmacogenetics of platinum-based chemotherapy in Non Small Cell Lung Cancer (NSCLC) roma patients. A trial was conducted to determine the association between genetic polymorphisms and platinum-based chemotherapy by checking odds ratio (OR) and 95% confidence interval (CI) in 39 roma patients treated in ontological clinic of University Hospital Tirana Center “ Mother Teresa”, between 2008-2012. Data were extracted from this trial study, and showed 11 polymorphisms in 8 genes for meta-analysis. MDR1 C3435T (OR = 1.97, 95% CI: 1.11–3.50, P = 0.02), G2677A/T (OR = 2.61, 95% CI: 1.44–4.74, P = 0.002) and GSTP1 A313G (OR = 0.32, 95% CI: 0.17–0.58, P = 0.0002) were significantly correlated with platinum-based chemotherapy in roma NSCLC patients.

Conclusion Attention should be paid to MDR1 C3435T, G2677A/T and GSTP1 A313G for personalized chemotherapy treatment for NSCLC patients in roma population in the future.

RAD51 G135C polymorphism associated with recurrence risk in post-menopausal women with advanced ovarian cancer

Nogueira Augusto, Assis Joana, Gomes Mónica, Catarino Raquel, Abreu Miguel, Luís Michael, Pereira Deolinda, Medeiros Rui,

Introduction Ovarian cancer (OC) is the third most common gynecological cancer among women worldwide. In Europe, OC is the fifth most incident cancer in women but the main cause of death among the gynecological tumors. Unfortunately more than two thirds of patients have advanced disease at diagnosis. OC incidence is age related and is a characteristic of postmenopausal women. RAD51 is a crucial enzyme in DNA repair by homologous recombination (HR) and has been shown to interfere with the prognostic of patients treated with chemotherapy. Genetic polymorphisms in DNA repair genes seem to determine the overall DNA repair capacity, which in turn may affect the treatment response. We conducted this study to show the possible influence of the RAD51 G135C polymorphism (1801320) in damage repair capacity and disease-free survival in advanced ovarian cancer patients. Material and Methods: We analysed the RAD51 G135C polymorphism by PCR-RFLP in genomic DNA isolated from peripheral blood of 110 patients with advanced ovarian cancer (FIGO stage III and IV) submitted to chemotherapy. Disease-free survival was the endpoint of this analysis and was defined as the time since end of treatment to recurrence or relapse. The associations between RAD51 G135C genetic variants and disease free interval were estimated by Kaplan Meier methodology and using Log Rank test.

Results Our results demonstrate that the time to first recurrence was statistically different according to the patients RAD51 G135C genotypes. The patients carrying the C-allele present a more recurrence risk than patients with GG genotypes (P=0.004). Cox regression analysis adjusted by stage disease, age, family history, lymphatic invasion, venous invasion and presence of ascites confirmed this association, indicating that patients carriers of the variant C allele present an increased risk of 2.1-fold for the recurrence risk [Hazard Ratio (HR), 2.11; 95% CI, 1.01-4.42; P=0.046; Pbootstrap=0.036].

Conclusions This is first study evaluating the possible influence of the RAD51 G135C polymorphism in recurrence risk of advanced ovarian cancer. Our results indicate that of the RAD51 genetic variants influence of disease-free survival, conferring an increased of recurrence risk of these patients. These results may contribute to a better understanding of the role DNA repair polymorphisms in recurrence risk of advanced ovarian cancer and treatment response in these patients.

Development of One-step DNA Chip for VKORC1 and CYP2C9 genotyping

Park Seungman, Moon Hee-Won, Lee Do-Bu, Seo Sung-Min, Ku Su-Jin, Paek Se-Hwan, Paek Moon-Cheol, Yun Yeo-Min

Warfarin is the most commonly used oral anticoagulant but also is the second leading cause of emergency room visits for adverse drug reactions. Starting of warfarin therapy is complicated by many factors, including a narrow therapeutic range and substantial genetic variability between patients. There are several methods (Real-Time PCR, DNA Chip and DNA Sequencing) for genotyping of warfarin related genes. But long time consumption and high cost is not suitable for cost-effective and convenience detection. Because these reason we have developed rapid and economic one-step DNA chip for genotyping of the VKORC1 and CYP2C9 genes. And we validated analytical performance of DNA chip in comparison to those of real-time PCR and direct sequencing. We designed a DNA chip devices carrying a multi-stage well to perform PCR and hybridization. Probes for identifying genotypes were immobilized on the surface of DNA chip. Clinically important genotypes were included such as CYP2C9 *2 (3608C>T), CYP2C9 *3 (42614A>C), CYP2C9 *5 (42619C>G), CYP2C19 *17 (99C>T), and VKORC1 (6399C>T). After on-slide PCR and hybridization, spot signals were analyzed with fluorescence scanner. The results from our device were compared with those of real-time PCR and direct sequencing. DNA chip tests showed same results with real-time PCR and direct sequencing analysis. In conclusion, one-step DNA chip can replace the real-time PCR and DNA sequencing for SNP analysis of Warfarin dose decision. Furthermore, since one-step DNA chip can analyze diversity of genotypes and larger numbers of samples simultaneously compare to current methods, we can expect one-step DNA chip to generate more accurate results in more economical manner.

A case report of voriconazole therapeutic drug monitoring in a heterozygous ultrarapid CYP2C19*1/*17 patient

Quaranta Sylvie, Scodavolpe Simon, D’Incan Corda Evelyne, Saut Noémie, Berger Pierre, Morange Pierre-Emmanuel, Lacarelle Bruno, Solas Caroline

Invasive pulmonary aspergillosis (IPA) is a major cause of both morbidity and mortality in neutropenic patients. Voriconazole (VRZ) is a triazole antifungal agent active in acute invasive aspergillosis. VRZ is extensively metabolized by the cytochrome P450 system including CYP2C19, CYP2C9 and CYP3A. Thus, polymorphisms in the CYP2C19 gene have substantial impact on the pharmacokinetics of VRZ. Especially, subjects carrying a CYP2C19*17 allele may be at higher risk of sub-therapeutic VRZ concentration, and subsequently of treatment failure. We report the case of a patient with an IPA failing on VRZ therapy harboring a CYP2C19*17 variant, associated with a low VRZ trough concentration. A 51-year-old Caucasian woman treated for acute myeloid leukemia presented a right chest pain without any other clinical symptomatology. Multiple nodular lesions on the high-resolution CT scan allowed to suspect an IPA. Detection of aspergillus antigen in pulmonary nodular biopsy confirmed the diagnosis. Oral VRZ was started at 200 mg twice-daily. Therapeutic drug monitoring of VRZ was performed as recommended and showed a sub-optimal VRZ trough serum concentration at 0.38 µg/mL. Co-medications impacting VRZ exposure and absorption disorders were checked. A CYP2C19 ultrarapid metabolism was suspected regarding both the sub-optimal VRZ concentration and the radiological non-response 3 weeks after initiation of VRZ. CYP2C19 genotyping was performed and revealed a heterozygous ultrarapid CYP2C19*1/*17 genotype. Consequently, the VRZ dose was increased to 300 mg twice-daily and the VRZ trough serum concentration reached 2.2 µg/mL six day after. This observation report an association between CYP2C19*1/*17 genotype, sub-optimal VRZ exposure and treatment non-response. The dose adjustment of VRZ allowed to achieve VRZ concentration within the therapeutic range (1 to 4 µg/mL) and a regression of the lesions on the CT scan was observed 3 weeks later. To date, VRZ exposure in CYP2C19*17 ultrarapid metabolizers has not been clearly described yet. The interest and indications of CYP2C19 genotyping along with VRZ therapeutic drug monitoring require further clinical investigation.

Impact of P450 oxidoreductase (POR) *28 allele on lipid lowering response to atorvastatin and simvastatin in children and adolescents with familial hypercholesterolemia and adults with primary hypercholesterolemia

Ragia Georgia, Drogari Euridiki, Kolovou Vana, Elens Laure, Mollaki Vicky, Tavridou Anna, Van Schaik Ron, Kolovou Genovefa, Manolopoulos Vangelis

Background and Aim Statins are effective agents for lowering LDLc in patients with familial (FH) and/or primary (PH) hypercholesterolemia. Interindividual variation in response to statin therapy is partially attributed to genetic factors. The polymorphic enzyme P450 oxidoreductase (POR) transfers electrons from NADPH to CYP3A enzymes, which metabolize atorvastatin and simvastatin. POR*28 polymorphism is associated with increased activity of CYP3A enzymes. We analyzed the association of POR*28 allele with response to statins.

Methods Three statin-treated populations were studied, 106 atorvastatin treated children and adolescents clinically diagnosed with FH, 207 atorvastatin treated adults with PH and 210 simvastatin treated adults with PH. Total cholesterol (TChol) and LDLc were measured at baseline and after 6 months of treatment. POR*28 allele was analyzed with TaqMan assay.

Results In FH population, POR*28 carriers had significantly lower % mean reduction from baseline of TChol (-33.1% in *1/*1, -29.8% in *1/*28 and -25.9% in *28/*28 individuals, p=0.045) and of LDLc (-43.9% in *1/*1, -40.9% in *1/*28 and -30.8% in *28/*28 individuals, p=0.013). In multivariable linear regression adjusted for confounding factors, POR*28 genotypes, additionally to baseline cholesterol level, account for an estimated 8.3% and 7.3% of overall variability in % TOTc and LDLc reduction (beta:4.05; 95% CI 1.73-6.37; p=0.001 and beta:5.08; 95% CI 1.62-8.54; p=0.004, respectively). In atorvastatin treated adults, POR*28 allele was associated with a non-significant trend towards lower % mean reduction from baseline of TChol (-35.5% in *1/*1, -33.7% in *1/*28 and -29.8% in *28/*28 individuals) and LDL-c (-42.6% in *1/*1, -41.7% in *1/*28 and -37.8% in *28/*28 individuals). This trend was not observed in the cohort of simvastatin-treated adults.

Conclusions In FH children, carriage of POR*28 allele was associated with reduced effect of atorvastatin on TChol and LDLc and identified FH children that may require higher atorvastatin doses to achieve full therapeutic benefits. In adults, POR*28 allele was not associated with lipid lowering to atorvastatin and the results were replicated in an independent simvastatin-treated population. In adult patients, the hypothesized effect of POR*28 allele on lipid lowering response to CYP3A metabolized statins can potentially be masked by relevant confounding or uncontrolled factors.

Association of CYP2C9*2 and *3, POR*28 and KCNJ11 E23K gene polymorphisms with hypoglycemia in sulfonylurea-treated type 2 diabetic patients

Ragia Georgia, Tavridou Anna, Van Schaik Ron, Manolopoulos Vangelis

Background: The therapeutic response to sulfonylureas and the incidence of hypoglycemic episodes in T2DM patients treated with these drugs are influenced by patients co-medication, co-morbidity, age, sex, liver and renal function, as well as by genetic factors. Changed pharmacokinetics or pharmacodynamics of sulfonylureas due to polymorphisms in CYP2C9 and POR enzymes that are involved in their metabolic pathway and in KCNJ11 gene that encodes the inward rectifier K+ channel Kir6.2 of the KATP channel that sulfonylureas block in pancreatic β-cells, may partially explain the differences in patient dose requirements and frequency of adverse reactions.

Aim To assess the association of CYP2C9*2 and *3, POR*28 and KCNJ11 E23K polymorphism with sulfonylurea-induced hypoglycemia.

Methods 92 T2DM patients receiving sulfonylurea and reporting drug-associated hypoglycemia and 84 T2DM patients receiving sulfonylurea and having never experienced hypoglycemia were included in the study. CYP2C9*2 and *3, POR*28 and KCNJ11 E23K polymorphism were detected by use of PCR‐RFLP and TaqMan methods.

Results CYP2C9*1/*3 genotype was associated with sulfonylurea-induced hypoglycemia, while no differences in CYP2C9*2, POR*28 and KCNJ11 E23K allele frequency between the two groups were found. In an adjusted model, CYP2C9*1/*3 genotype increased the hypoglycemia risk in response to sulfonylurea (odds ratio: 1.687; p=0.011). Because POR*28 increases CYP2C9 activity and may masque CYP2C9*2 effect on sulfonylurea metabolism, the association of CYP2C9*2 allele with hypoglycemia was further investigated in POR*1/*1 individuals. In POR*1/*1 patients, CYP2C9*1/*2 genotype was more common in cases than in controls (p=0.041). In an adjusted model, CYP2C9*2 allele was also associated with increased risk of hypoglycemia (odds ratio: 3.218; p=0.031).

Conclusions In T2DM patients, KCNJ11 E23K polymorphism is not associated with increased risk of mild hypoglycemia in sulfonylurea-treated T2DM patients. CYP2C9*3 increases the risk of hypoglycemia possibly due to impaired metabolism of these drugs. In the absence of POR*28 allele, the presence of CYP2C9*2 allele is also a major determinant predisposing sulfonylurea-receiving patients to hypoglycemia. CYP2C9 and POR polymorphisms genotyping might thus be useful tools for predicting adverse effects caused by sulfonylureas and help clinicians in safer prescribing of oral hypoglycemic agents.

Combined effects of OCT1 and CYP2D6 on uptake and metabolism of debrisoquine

Saadatmand Ali Reza, El-Armouche Ali, Brockmöller Jürgen, Tzvetkov Mladen

Debrisoquine is a model substrate of CYP2D6 that played a pivotal role in the history of pharmacogenetics. Recently we showed that debrisoquine is a substrate of the organic cation transporter OCT1. OCT1 is strongly expressed in the human liver and may contribute to debrisoquine metabolism by mediating debrisoquine uptake in hepatocytes. In this study, we established HEK293 cells co-overexpressing OCT1 and CYP2D6 to analyze OCT1-CYP2D6 interactions during the metabolism of debrisoquine. We analyzed whether OCT1 overexpression increase debrisoquine metabolism and whether genetic polymorphisms in OCT1 gene or drug–drug interactions at OCT1 affect debrisoquine metabolism.

OCT1 overexpression significantly increased the production of 4-OH debrisoquine, the main metabolite of debrisoquine, in HEK293 cells overexpressing CYP2D6. The effect was time dependent. The intracellular concentrations of 4-OH debrisoquine was more than 3-fold higher in the first 20 min, but did not differ after 90 min of debrisoquine exposure between the cells co-overexpressing OCT1 and CYP2D6 and cells overexpressing CYP2D6 alone. Loss-of-function genetic polymorphisms in OCT1 reduced both the uptake of debrisoquine and the production of 4-OH debrisoquine.

We also analyzed the effect of drug-drug interaction on the uptake and metabolism of debrisoquine using paroxetine, quinidine and ritonavir. Paroxetine and ritonavir were equally potent in inhibiting the uptake of debrisoquine and the metabolism to 4-OH debrisoquine. The IC50 of paroxetine were 1.96 ± 0.368 µM and 1.22 ± 0.6 µM for the uptake and the metabolism of debrisoquine, respectively. The IC50 of ritonavir were 4.87 ± 3.43 µM and 7.07 ± 2.91 µM for the uptake and the metabolism, respectively. In contrast, quinidine was much more potent in inhibiting the metabolism (IC50 = 0.335 ± 0.045 µM) than the uptake of debrisoquine (IC50 = 22.06 µM).

In conclusion, OCT1 may limit debrisoquine metabolism via modulating the access of debrisoquine to CYP2D6. This work showed also that cell models co-overexpressing OCT1 and CYP2D6 are useful tool for understanding the interplay between uptake and metabolism of drugs and analyze epistatic effects of polymorphisms and complex drug-drug interactions.

Statins deregulate microRNA expression signature in HepG2 cells

Salazar Luis, Zambrano Tomás, Hirata Mario, Cerda Alvaro, Hirata Rosario

Hypercholesterolemia is an important risk factor for cardiovascular disease. Statins are the cholesterol-lowering drugs of choice widely used to reduce plasmatic cholesterol levels in cardiovascular patients. The present study aimed to investigate the effect of statins therapy on the expression pattern of microRNAs using an in-vitro model of liver hepatocellular cells (HepG2) submitted to therapy with 2 different statins. Materials and Methods: microRNA expression profiling of HepG2 cells treated with 2 different statins was carried out. Expression was examined using a microarray chip containing 2019 probes. Transcripts are listed in Sanger miRBase Release 19.0. Normalization was carried out using LOWESS (locally weighted scatterplot smoothing) method. Potential miRNAs regulating target genes and their preliminary biological functions are up to undergo qPCR analysis for validation and function prediction software.

Results When using simvastatin therapy, HepG2 cells showed a total of 9 miRNA differentially expressed, whereas for atorvastatin therapy, the expression pattern of 35 microRNAs was deregulated. Two miRNA (hsa-miR-4485 and hsa-miR-6510-5p) were significantly differentially expressed between the two groups.

Conclusion MicroRNA profiling successfully detected a branch of differential expression miRNAs between 2 different hypolipemiant therapies. Of these, Atorvastatin showed a more potent effect on microRNA signature, affecting more transcripts than simvastatin. Also, 2 microRNAs were positively deregulated simultaneously in both treatments. Further qPCR analysis and biological function analysis are needed to explore comprehensively these findings.

Influence of GEMIN4 rs7813 (T>C) functional polymorphism in time to castration-resistance in prostate cancer patients submitted to androgen deprivation therapy

Santos Juliana Inês, Teixeira Ana L, Dias Francisca, Gomes Mónica, Nogueira Augusto, Assis Joana, Lobo Francisco, Morais António, Maurício Joaquina, Medeiros Rui

Background Prostate cancer (PC) is an important public health problem, being the malignant neoplasia with the highest incidence rate in men in developed countries. Androgen deprivation therapy (ADT) is commonly used as the primary therapy for patients diagnosed in advanced stages. However, statistical data has shown that use of ADT only slightly improves the overall survival (OS) and disease usually progresses to castration resistant prostate cancer (CRPC). The CRPC is associated with tumor progression, metastatic disease development, acquisition of an aggressive PC phenotype and actually with no effective therapies. Recently, microRNAs (miRNAs) have been defined as a new class of regulatory genes that modulate the gene expression by binding to its target messenger RNA (mRNA). Many cellular and physiological processes in health and disease were associated with changes in miRNAs expression. Increasing evidence suggests that miRNAs are involved in cancer development and progression, due to changes in regulation of miRNAs processing. The Gem-associated protein 4 (GEMIN4) belongs to a complex of four molecules – RISC complex- involved in binding of miRNAs to mRNAs. Recently, a T>C transition in GEMIN4 gene was described, in which, the C variant was associated in vitro with cellular growth inhibition. Our purpose was to investigate the potential influence of the GEMIN4 rs7813 (T>C) functional polymorphism in acquisition of CR phenotype in response to ADT. Material and Methods: A follow-up study (100 months) was undertaken to evaluate response to ADT, in 295 patients, with histopathologically confirmed PC. Genotyping of GEMIN4 rs7813 (T>C) genetic polymorphism was performed by Real-Time PCR allelic discrimination method.

Results Concerning the time to CR after the beginning of ADT, we observed a significantly reduced time-to-CR in GEMIN4 rs7813 TT/TC carriers compared with CC genotype carriers (55.6 versus 70.3 months) (Breslow test, P=0.043). Furthermore, multivariate Cox regression model using tumor stage, Gleason≥ 8, and PSA levels at diagnosis as co-variants, demonstrated a higher risk of an earlier relapse in GEMIN4 rs7813 TT/TC genotypes patients carriers than CC carriers following ADT (hazard ratio- HR= 1.87, 95%CI: 1.08-3.20, P= 0.024).

Conclusions Our results suggest that GEMIN4 rs7813 TT/TC genotype may contribute to an early CR phenotype acquisition in patients submitted to ADT. Thereby, GEMIN4 rs7813 T>C genotypes may help to the establishment of a genetic profile to ADT efficacy in CRPC and support the impact of deregulation of miRNAs processing in PC progression.

Pharmacogenetic study of acute lymphoblastic leukemia

Semsei Agnes, Lautner-Csorba Orsolya, Kutszegi Nora, Sagi Judit, Falus Andras, Kovács Gabor, Szalai Csaba, Erdélyi Daniel J.

Nowadays the treatment of ALL is very effective, the vast majority of children with ALL gets cured for the long term. The survival rate improved remarkably over the last decades, now the 5-year survival rate is around 90%. But the used very effective chemotherapeutic agents have side effects. In our research our aim was to study acute and late toxic side effects of chemotherapy and to determine the genetic background of these side effects. Our study population constisted of 454 childhood acute lymphoid leukemia (ALL) patients of Hungarian (Caucasian) origin treated according to the ALL Berlin-Frankfurt-Münster (BFM) 90, 95 and / or ALL IC-BFM 2002 chemotherapy protocols. Patienst were genotyped for 189 single nucleotide polimorphisms (SNP) in 45 genes coding transporters and metabolizing enzimes of chemotherapeutic agents. Clinical data of toxicity regarding antracycline induced cardiotoxicity, acute central nervous system (CNS) complications of grade II or above and asparaginase allergy were collected retrospectively from the patiens medical records. The data were evaluated with traditional frequentist-based methods and we applied a new statistical method, called Bayesian network based Bayesian multilevel analysis of relevance (BN-BMLA) to study interactions of SNPs. Using traditional frequentist based method association of grade II-IV neurotoxitiy was found with ABCC1 rs246219 CC genotype (OR=5.1, CI 95%=1.15-25, p=0.032) and with MVP rs4788186 G allele (OR=0.38, CI 95%=0.17-0.88, p=0.024). The population is also searched for potential interaction the studied SNPs with BN-BMLA analysis, but it is still in progress on our dataset. Our results indicate that genetic variations in genes involved in drug resistance may have impact on the acute CNS toxicity of chemotherapy. SNPs can be used as genetic markers to identify patients at higher risk of toxicity. This approach has a potential for individualizing therapy in the future.

Ara-C induced toxicity: A deadly gift

Serdjebi Cindy, Ciccolini Joseph, Fina Frédéric, Verschuur Arnaud, Ouafik L’Houcine, André Nicolas

Cytosine arabinoside (Ara-C) is an antimetabolic agent used to treat different forms of hematologic cancers, including Burkitt lymphoma. As a pyrimidine analogue, its deactivation in uracil-arabinoside is mainly dependant on cytidine deaminase (CDA), a liver enzyme that proved to be highly polymorphic. This polymorphism, partly explained by genetic causes, leads to an activity is ranging from poor metabolizer (PM) to rapid deaminator (UM).

We reported here a very rare patient case of a young female child, treated with Ara-C for a Burkitt lymphoma. In April 2012, clinicians diagnosed an EBV induced-lymphomatous cell proliferation. Before starting Ara-C treatment, the patient received rituximab, cyclophosphamide, vincristine and doxorubicin without having any toxicity. After a preventive routine-test which consists in evaluating cytidine deaminase residual activity in serum, we detected a CDA deficiency (2.6 vs 3.5 U/mg of proteins in general population). Despite a 30% decrease of Ara-C dosage, the patient declared severe hematological toxicities, such as febrile aplasia, septic shock, hemodynamic disorders, and neutropenic colitis.

By collecting patient data, we noticed that this patient underwent liver transplantation after biliary atresia in her early childhood. As cytidine deaminase is produced by liver, we hypothesized that CDA deficiency may be the result of a genetic polymorphism from the donor graft. We collected paraffin sections from the donor and peripheral blood from the patient to extract DNA and performed a genotyping test. Preliminary results suggest that patient was wild-type for c79A>C, -31delC, c208G>A. However, it appeared that donor DNA was homozygous for c79A>C, a polymorphism which may be responsible for a decreased in CDA activity. Sequencing is currently underway to confirm those findings.

This case report confirms that the routinely used test is appropriated to detect a CDA deficiency from a serum sample as a predictive marker of liver CDA activity. It also illustrates whether it is important to preventively screen for patients at risk of toxicity before a pyrimidine analogue-based treatment.

Cytidine deaminase deficiency and severe toxicities upon azacytidine intake: Clinical observations and experimental evidences

Serdjebi Cindy, Fanciullino Raphaëlle, Ciccolini Joseph, Mercier Cédric, Costello Régis, Lacarelle Bruno, Ouafik L’Houcine

Azacytidine is a nucleosidic analog used for treating a variety of haematological malignancies. Azacytidine pharmacokinetics is dependent upon a liver detoxification step driven by cytidine deaminase (CDA). CDA is affected by several genetic polymorphisms leading to a wide inter-individual variability in resulting enzymatic activities. In particular, dysregulated CDA has been associated with life-threatening toxicities with several nucleosidic analogs such as cytarabine, capecitabine and gemcitabine intake. However, little is known about the impact of CDA deficiency on the clinical outcome of patients treated with azacytidine. In our institute, a lethal toxicity after pancytopenia has been observed with an 82-years old female patient upon azacytidine intake. CDA deficiency was evidenced after retrospective phenotyping (functional activity was found to be reduced by 65% as compared with standard CDA values: 1.2 VS. 3.5 U/mg), thus strongly suggesting that impaired ability to detoxify azacytidine could be the culprit indeed for this case. To further establish the exact role CDA-deficiency could have played in this toxic-death, a non-clinical study was undertaken in rodents. To mimic CDA deficiency, 8 mice were pre-treated with 100 mg/kg of tetrahydrouridine I.P. before being administered with 24 mg/kg of azacytidine. Another group of 8 mice received 24 mg/kg azacytidine only, and were therefore considered as control-CDA. Four mice were used as untreated controls when evaluating tolerance to the treatment. Neutrophils were monitored by flow-cytometry before and after treatment over 10 days as a surrogate marker for global azacytidine-related toxicities. CDA was measured using the same test than the one used at bedside and showed that the two groups differed markedly in their CDA phenotypes (ie, 7.7 VS. 0.98 U/mg) and that deep CDA deficiency (Poor Metabolizer: PM status) was achieved indeed in THU-treated animals. Pharmacokinetics study was carried out on a satellite group (T10, T20, T40 and T60 min) by revered-phase HPLC-UV analysis and showed a marked overexposure in azacytidine plasma levels in PM animals displaying CDA-deficiency. In full line with this observation, longer and deeper neutropenia was observed in this PM subset, as compared with the mice with normal CDA activity treated with the same dosage of azacytidine. Overall, our experimental data strongly support the hypothesis that CDA deficiency is a condition associated with increased risk to undergo life-threatening toxicities after azacytidine treatment. Detecting PM patients should help to secure the use of azacytidine at bedside.

Genetic-determined low response to thienopyridines is associated with higher systemic inflammation in smokers

Shahabi Payman, Cuisset Thomas, Herbeth Bernard, Morange Pierre Emmanuel, Grosdidier Charlotte, Stathopoulou Maria, Siest Gérard, Alessi Marie-Christine, Visvikis-Siest Sophie

Aims Cytochrome P450 (CYP) 2C19 loss- and gain-of-function polymorphisms (CYP2C19*2 and CYP2C19*17) influence the response to the anti-platelet effects of both clopidogrel and prasugrel. Also, some, but not all, studies suggested that smoking positively modify response to clopidogrel, but not prasugrel. Moreover, post-procedural CRP level is a reliable predictor of post-PCI complications like in-stent restenosis. This study sought to investigate whether the interactions of *2 and *17 with smoking are associated with the levels of P2Y12 receptor inhibition and C-reactive protein (CRP), in patients undergoing percutaneous coronary intervention (PCI) treated with aspirin and either clopidogrel or prasugrel.

Methods and results In total, 1128 patients with acute coronary syndrome who underwent a successful PCI were recruited. At one-month clinical follow-up, the interactions of smoking and CYP2C19 polymorphisms on the levels of on-treatment platelet reactivity, assessed by vasodilator-stimulated phosphoprotein platelet reactivity index (VASP PRI) and CRP were explored in three metabolizing groups as follow: poor metabolizers (PM) (*2 carriers/*17 non-carriers); intermediate metabolizers (IM) (*2 carriers/*17 carriers or *2 non-carriers/*17 non-carriers); and ultrametabolizers (UM) (*2 allele non-carriers/*17 carriers). There was a significant difference in VASP PRI, but not CRP, levels between the three metabolizing groups (p < 0.001 and p = 0.412, respectively). The interactions of metabolizing status and smoking was significant for CRP (p = 0.004) but not for VASP PRI (p = 0.720).

Conclusion Interaction between CYP2C19 polymorphisms and smoking modifies the on-treatment CRP level of post-PCI, on-thienopyridine patients. This effect seems to be independent to the level of P2Y12 receptor inhibition.

In silico analysis of SNPs in pharmacogenomic studies: predictive functional analysis of SLC5A5 gene variants and potential impact on radioactive iodine (131-I) therapy response

Silva Santos Luis

Single nucleotide polymorphisms (SNPs) are a common type of genetic variation that may be located in coding or non-coding regions of the DNA double-strand. The vast majority of these variants are devoid of functional significance but some have the potential to alter the corresponding protein’s structure, function or expression and can thus influence individual disease susceptibility, drug response or chemical exposure sensitivity. Experimental methods to distinguish functional from neutral SNPs are challenging, though. Therefore, given the vast amount of SNPs with unknown functional significance that exists throughout the human genome, predictive a priori identification of potentially functional SNPs through computational methods could prove useful to prioritize candidate variants prior to experimental functional analysis and genetic association studies. Such in silico approach could also be applied to the post hoc evaluation of variants identified in whole genome scans or within haplotype blocks associated with disease or drug response, ultimately yielding new genetic susceptibility biomarkers.

Post-thyroidectomy radioactive iodine (131-I) therapy has long been used in the management of patients with well-differentiated thyroid cancer of follicular cell origin (papillary or follicular thyroid cancer). Unfortunately, a highly significant fraction of thyroid cancers are either resistant or become resistant to 131-I. Radioiodine induces cell death by emission of short path-length radiation and must, therefore, be taken up by thyroid follicular cells to be effective. Cellular uptake of radioiodine is mediated through a membrane transporter, the sodium/iodide cotransporter, primarily expressed in thyroid tissue where its main function is to promote iodide uptake. This transporter is encoded by the SLC5A5 gene. SLC5A5 mutations are associated with thyroid dyshormonogenesis 1, a disorder characterized by iodide transport defects leading to congenital hypothyroidism. Also, SLC5A5 expression is reduced in thyroid tumors, a factor that could account for low 131-I uptake (hence resistance) in thyroid cancer tissue. Therefore, it is possible that SLC5A5 functional variants influencing the function or expression of the sodium/iodide cotransporter may interfere with the ability of thyroid follicular cells to uptake 131-I and thus impact the efficacy of radioiodine therapy.

In this work, through the combined use of several in silico computational methods such as Sorting Intolerant from Tolerant (SIFT), Polymorphism Phenotyping (PolyPhen) and FASTSNP, we aimed to predict the phenotypical impact of SNPs on the SLC5A5 gene, so that the most probable functional SNPs are identified and prioritized for subsequent experimental studies. The predicted results were then validated by phenotypical data reported in prior functional and genetic association studies, when available.

Based on this approach, a comprehensive set of SLC5A5 variants were predicted to have functional consequences: some of these have been previously validated through prior studies, reinforcing the value of this approach, while other potentially deleterious variants are added, providing useful hints for future studies on the interindividual variation in response to 131-I therapy. Concluding, the bioinformatic approach proposed here integrates relevant biomedical information sources to provide a systematic genotype-phenotype analysis that can prove useful prior to experimental pharmacogenomic association studies.

CYP2B6 haplotype and biological factors responsible for hepatotoxicity in HIV-infected patients receiving efavirenz-based antiretroviral therapy

Sukasem Chonlaphat, Manosuthi Weerawat,Lueangniyomkul Aroon, Mankatitham Wiroj, Thongyen Supeda, Nilkamhang Samruay, Manosuthi Sukanya, Sungkanuparph Somnuek

Data on the pharmacogenetic marker of CYP2B6 and biological factors which were associated with hepatotoxicity in HIV-infected patients receiving efavirenz-based antiretroviral regimen is very limited. A total of 134 HIV-infected Thai adults were prospectively enrolled to receive a once daily regimen of efavirenz 600 mg/tenofovir/lamivudine. Seven single nucleotide polymorphisms (SNPs) within CYP2B6 were genotyped using real-time PCR. At 12 weeks after ART, plasma efavirenz concentrations at 12 hours after dosing and liver chemistries were measured. Of all, mean±SD age was 37±8 years and 78% were male. The median (IQR) CD4 count was 43 (17–105) cells/mm3. Eighteen (13%) patients had positive anti-HCV and 5 (4%) had positive HBsAg. The frequencies of wild type and heterozygous/homozygous mutant of each SNP were 64C>T (89%, 11%), 499C>G (100%, 0%), 516G>T (45%, 55%), 785A>G (37%, 63%), 1375A>G (100%, 0%), 1459C>T (96%, 4%), and 21563C>T (38%, 62%). Three most frequent haplotypes identified included *1/*6 (40%), *1/*1 (34%), and *6/*6 (8%). Median (IQR) plasma efavirenz concentration was 2.3 (1.4-3.7) mg/L. At 24 weeks, median (IQR) serum ALP was 98 (73-133) mg/dL, direct bilirubin was 0.11 (0.10-0.19) mg/dL. The proportion of grade 1 and grade 2 elevated serum ALP were 12.7% and 1.5%, respectively. By multivariate analysis, factors associated with high ALP, total bilirubin, and direct bilirubin at week 24 included CYP2B6 haplotype *6/*6, high serum ALP at week 0, and positive anti-HCV (all P<0.05). In summary, HIV-infected Thai patients who have pharmacogenetic marker“CYP2B6 haplotype *6/*6” may increase susceptibility to hepatotoxicity with efavirenz-based ART.

A prospective study of the effectiveness of pharmacogenetic approach to the warfarin appointment in a Russian hospital

Sychev Dmitry, Karasev Alexandr, Belyakov Ilya, Lokhonina Anastasia, Otdelenov Vitaliy, Kleimenova Elena, Nazarenko Gerasim

The influence of polymorphisms CYP2C9 and VKORC1 genes, the amount of the maintenance dose of warfarin anticoagulation stability and development of haemorrhagic complications were studied in the retrospective studies mainly. The aim of this study was to evaluate the effectiveness of pharmacogenetic approach to warfarin dosing over traditional in a versatile hospital. A prospective study was performed in the cardiological, therapeutic, surgical departments and intensive care unit for the period 2011 – 2012 year. The main group enrolled 130 patients (72 men and 58 women) at the age of 68,8 ± 12,2, with the primary warfarin dose of 5 mg/day on indications: atrial fibrillation (n = 121), phlebothrombosis (n = 5), heart valve replacement (n = 4). Polymorphisms G3673A in VKORC1 gene, CYP2C9*2, CYP2C9*3, associated with increased sensitivity to warfarin were assessed by real-time PCR. The result of the study, and carried out on the basis of its calculation of the maintenance dose according to the protocol Gage (2009) (using on-line calculator www.warfarindosing.org)physicians received on the second day, and then adjust the dose of warfarin, further dose adjustment was carried out on the INR value. The control group enrolled 80 patients in whom warfarin was administered at a dose of 5 mg/day, and dose selection was carried out on the basis of the determination of the INR only. The primary end-points were the warfarin dose titration differences in the number of steps were performed and the number of patients with excess of INR greater than 3 during the 6-month follow-up period (except the heart valve replacement patients).

Results In the main group identified alleles increased sensitivity to the VKORC1 gene in 57.6% (genotypes AG and AA), in the gene CYP2C9 (genotypes of CYP2C9 *1/*2, *1/3, *2/*3, *3 /*3) - 32.3%, with 17.7% of patients had a combination of VKORC1 and CYP2C9 alleles. Given the results of pharmacogenetic testing, the estimated maintenance dose of warfarin in 59.7% of the treatment group was less than 5mg/day, in 10.9% recommended doses were less than 2.5 mg. The number of steps of dose titration of warfarin to a target INR level was 3,3 ± 1,2 in the main group and 5,8 ± 2,7 in the control group (p = 0.034). The number of patient with INR excess in the main group was 7%, in the control group 20% (p=0.022). The correlation between the calculated and selected maintenance doses of warfarin in the study group estimated as r = 0,77, p = 0.02. Haemorrhagic complications in the main group weren’t observed for the 6-month after warfarin initiation, one patient of the control group revealed bleeding from intestinal polyp.

Conclusion The study suggests that pharmacogenetic approach to warfarin dose adjustment in versatile hospital is more effective then traditional one.

Possibilities of warfarin metabolism enzyme CYP2C9 analysis for safe patients’ anticoagulant therapy after the cardiac valve replacement

Sychev Dmitry, Arslanbekova Serminas, Kuznetsova Elena, Goluhova Elena, Kazakov Ruslan

Aim Estimate the influence of gene CYP2C9 polymorphism activity on maintenance dose of warfarin for patients after the cardiac valve replacement. Materials and methods: 33 patients with replaced cardiac valves were examined, their average age was 45,5±13,8 years. Before the replacement of cardiac valves all patients’ СYP2C9 genotype carrier were identified by PCR-RFLP method after DNA preallotment from the whole blood. After one-time administration of 50 milligrams of losartan CYP2C9 activity was evaluated by losartan metabolite concentration (Е-3174) in urine that was taken during eight hours. The concentration of active metabolite Е-3174 was evaluated by high-efficiency liquid chromatography method.

Results Warfarin doses in early and late (after 6-12 months) postoperative patients’ period were compared depending on concentration of active metabolite Е-3174 (more or less than 2500 UFH/ml). In the case of concentration of metabolite more than 2500, patients often were having dose of warfarin less than 5milligrams per 24 hours in comparison with patients, who had the metabolite concentration more than 2500 UFH/ml: 83% vs 20%, р=0,008. After division of patience in accordance with CYP2С9 gene polymorphism this difference remained unchanged with CYP2С9*1/*1 (66% vs 12%, р=0,049) genotype carriers, but not with the patients CYP2С9 не*1/*1, р=0,485 genotype carriers. Metabolite Е-3174 concentration in urine is less than 2500 UFH/ml according to the results of losartan test, that was made before the surgery, with responsivity 83% and specificity 80% forecasts the result in “small” doses of warfarin (less than 5 milligrams) in late postoperative period, with the prognostic value of positive effect 83% and prognostic value of negative effect 80% [OR=20 ID95% 2,284-175, 13]. Summary: Estimation of losartan active metabolite in urine would help to forecast the maintenance dose of warfarin that may foster improvement of the effectiveness and safety of medical treatment of patients after the cardiac valve replacement.

Influence of TGFBR2-875G>A polymorphism in gene expression and castration-resistant prostate cancer development

Teixeira Ana L, Gomes Mónica, Nogueira Augusto, Assis Joana, Dias Francisca, Santos Juliana I, Lobo Francisco, Morais António, Maurício Joaquina, Medeiros Rui

Background Prostate cancer (PC) is the most frequently diagnosed cancer in men and the fifth cause of death by cancer. Androgen deprivation therapy (ADT) is frequently used in advanced stages, albeit most men will eventually fail this therapy and die from recurrent castration-resistant prostate cancer (CRPC). The CRPC treatment is limited, ineffective and the acquisition of castration-resistant (CR) phenotype is associated with the activation of signaling pathways mediated by growth factors. The TGFβ1 and its receptors have an important role in tumor progression, being the pro-apoptotic function modulated by the expression of TGFβRII and the inhibitory SMAD7. A G>A transition in the -875 promoter position of the TGFBR2 gene was reported and it may enhance transcription activity. Our purpose was to investigate the potential influence of the TGFBR2-875G>A (rs3087465) in the circulating expression levels of TGFβRII and this capacity to modulate the response to ADT. Material and Methods: TGFBR2-875G>A (rs3087465) polymorphism was studied by allelic discrimination using real-time polymerase chain reaction (PCR) in 428 PC patients submitted to ADT. The TGFBR2 and SMAD7 mRNA relative quantification were performed using TaqMan real-time PCR method.

Results We found that TGFBR2-875GG homozygous patients present lower expression levels of TGFBR2 mRNA compared to AA/AG genotype carriers (AA/AG: 2-ΔΔCT =1.5, P=0.016). We also observed that GG genotype was associated with a higher Gleason grade (Odds Ratio- OR=1.51, 95%CI: 1.07-2.16, P=0.019) and with a significantly reduced time-to-CR compared with AA/AG genotypes carriers (86.7 versus 102.4 months) (Log Rank test, P=0.032). A multivariate Cox regression model using tumor stage, Gleason≥ 8, and PSA levels at diagnosis as co-variants, demonstrated a higher risk of earlier relapse in TGFBR2-875GG PC patients following ADT (hazard ratio- HR= 1.47, 95%CI: 1.05-2.05, P= 0.024). The concordance (c) index analysis showed an improvement of 6.3% in the prediction of CR development with the addition of TGFBR2-875G>A polymorphism information to a predictive model containing tumor clinicopathologic variables (0.746 versus 0.683).

Conclusions TGFBR2-875G>A contribution to an early relapse in ADT patients, due to changes in mRNA expression, supports the involvement of TGFβ1pathway in CRPC development. Furthermore, our results support the potential benefits of associate genetic information in predictive models of CR development.

The Turkish Oncology Group efforts to validate the applicability of various

Turhal Nazim Serdar, Yildiz Ramazan, Dane Faysal, Berk Veli, Cil Timucin, Kalender Mehmet Emin, Ozturk Akif, Gumus Mahmut

The Turkish Oncology Group efforts to validate the applicability of various chemotherapeutic doses and schedules in Turkish cancer patients The cancer treatment protocols including the dosing and schedule, overwhelmingly determined by the phase I-III studies designed in the developed world. The pharmocogenomic differences in countries outside of the developed world may make these dosing and schedule intolerable or ineffective. Toward this goal, several attempts carried out by the Turkish Oncology group to validate the applicability of various treatment protocols in Turkish cancer patients. These include, but not limited to: 1. The overall tolerated cumulative anthracycline dose of 144 breast cancer patients studied and the relative dose intensity rate was found to be 0.876 (042-1.06) which was consistent with the western literature. 2. The sunitinib tolerability and effectiveness studied in 102 renal cell cancer patients and although the rest of the side effects and survival data were comparable, close to 30 % of patients developed hypothyroidism that required treatment, which was unexpectedly high. 3. The tolerance of standard adjuvant treatment in 190 colon cancer patients studied and of the 5-fluorouracil (5-FU)-based chemotherapy group (n=141), 5% had a dose reduction because of toxicity and 73% were given the total planned dose and cycles, whereas these rates were 18.5 and 66% for oxaliplatin+5-FU treated group, respectively (p=0.66 and 0.44). 4. The 49 lung cancer patients treated with cisplatin based treatment studied for the toxicity and tolerance but a persistent toxicity or unexpectedly high intolerance is not observed. In conclusion, it is imperative for cancer specialists to study and report the treatment course of their own patients; develop management plans and allocate resources accordingly.

Methotrexate binds to recombinant thiopurine S-methyltransferase and inhibits enzyme activity after high-dose infusions in childhood leukaemia

Zimdahl Anna, Wennerstrand Patricia, Mårtensson Lars-Göran, Söderhäll Stefan, Lindqvist Appell Malin

6-mercaptopurine (6-MP) and methotrexate (MTX) are important drugs in the treatment of childhood acute lymphoblastic leukaemia (ALL). Thiopurine methyltransferase (TPMT) is a polymorphic enzyme causing variability in 6-MP response and toxicity. During ALL treatment, we have investigated the fluctuation in TPMT enzyme activity over time, as well as the effect of high-dose MTX infusions on TPMT enzyme activity and 6-MP metabolites. Fifty-three children with ALL treated according to the NOPHO-ALL 2000 protocol were included. TPMT enzyme activity was measured at six different times starting from diagnosis until after the end of maintenance treatment. TPMT and 6-MP metabolites were measured before and 66 hours after start of high-dose MTX infusions. The interaction between MTX and TPMT was investigated in vitro using recombinant TPMT protein and a leukemic cell line. We found that 40 percent of TPMT wild-type individuals had deceptively low TPMT enzyme activity compared to genotype at the time of diagnosis. TPMT decreased significantly 66 hours after the start of high-dose MTX infusions (-9.2 per cent, p=0.013) in the children. MTX bound to recombinant TPMT protein and inhibited the TPMT enzyme to a remaining activity of ~16 percent. Studies of thiopurine metabolism in cell lines further confirmed an inhibitory effect of MTX on TPMT activity. This study shows that TPMT genotyping should be preferred in children with ALL, since 40 per cent of TPMT wild-type children are at risk of initial under-dosing of 6-MP in cases where only TPMT enzyme activity is determined. MTX inhibits the TPMT enzyme activity after high-dose MTX infusions due to protein binding.

CYP3A4*22: A pharmacogenetic marker for CYP3A4 activity

van Schaik Ron, Elens Laure, Hesselink Dennis, Haufroid Vincent, Mathijssen Ron, de Graan Anne-Joy, Clarke Stephen, Charles Kelly, van Gelder Teun

Background: CYP3A4 is involved in the metabolism of 55% of all drugs. There are 21 DNA variants described for this gene, which, however, are not clinically significant because of low allele frequencies (<0.1%) or absence of a clear effect (CYP3A4*1B). In 2011, a new variant was described, CYP3A4*22 (intron 6 C> T (Wang et al 2011 Pharmacogenomics), which may be clinically relevant. Objective of our studies was to determine the effects of this variant on classic CYP3A4 phenotyping probes and examine correlations with four clinically used drugs.

Methods: The allele frequency was determined using TaqMan analysis in 6,285 Dutch individuals. For correlation with the CYP3A4 phenotype, 108 midazolam and 45 erythromycin treated patients we investigated. Effects on immunosuppressive therapy was studied in 357 kidney transplant patients (185x tacrolimus, cyclosporine 172x) and effects on paclitaxel therapy was studied in 239 oncology patients.

Results: CYP3A4*22 had an allele frequency of 6%, including 13 (0.2%) homozygotes. Midazolam concentrations were 32% higher (p<0.01) in CYP3A4*22 carriers while the metabolite formed by CYP3A4 in the erythromycin breath test was 40% lower (p = 0.032), both consistent with a reduced CYP3A4 activity. CYP3A4*22 carriers had a greater reduction in both total and LDL cholesterol, matching a lower CYP3A4 activity and thus higher levels of simvastatin. Tacrolimus dose requirement was 51% lower (p = 0.018) for CYP3A4*22 carriers to achieve similar trough levels. This effect was independent of CYP3A5 genotype. Cyclosporine-treated CYP3A4*22 carriers had a 20% lower creatinine clearance (p=0.002), matching increased on cyclosporine nephrotoxicity. Paclitaxel-related grade 3 neurotoxicity was significantly associated with CYP3A4*22 status (OR19.1, p=0.001).

Conclusions: with an allele frequency of 6% and significant effects on the metabolism of midazolam, erythromycin, simvastatin, tacrolimus, cyclosporine and paclitaxel, CYP3A4*22 has the potential of being a clinically relevant pharmacogenetic marker.

The TGFB1 Arg25Pro polymorphism and acute toxicity of radiotherapy: Studying the mechanism behind

Filonenko Kateryna, Brockmöller Jürgen, Schirmer Markus

Background: Two previous independent clinical studies comprising together 163 patients treated with neo-adjuvant radiotherapy for rectal cancer showed an association between a single nucleotide polymorphism (SNP) of the TGFB1 gene and the development of acute toxicity after radiotherapy (RT). This SNP causes the amino acid exchange Arg25Pro located in the signal peptide. All 21 patients carrying Pro25 experienced acute high grade treatment toxicity (HGAT). The aim of our study is to determine possible molecular mechanisms for the identified clinical association of the Arg25Pro polymorphism.

Methods: We set up for overexpression of Arg25 and Pro25 in a model system to investigate allele-specific effects when simulating radiotherapy. Therefore, we performed stable transfection of constructs for Arg25 and Pro25 in T-Rex HEK293 cell line using Flp-In system. The transfected cells were characterized on genome level by integration-specific PCR, on mRNA level by qRT-PCR, and on protein level by Western Blot. To detect the localization of TGFb1 protein immunocytochemistry was performed. FLAG tag-containing TGFB1 constructs with either Arg25 or Pro25 were generated for transient transfection. Functional effects were evaluated upon irradiation with 25 Gy. Amounts of intracellular and extracellular TGFb1 were measured by ELISA 24 and 48 hours after radiation and normalized to total protein by bicinchoninic acid assay.

Results: By ELISA, increased intracellular expression of TGFb1 was confirmed in T-Rex HEK293 cells for both the Arg25 and the Pro25 variant, which was accompanied by higher levels of secreted protein in relation to a transfectant control without TGFB1. When calculating the ratios between secreted and intracellular TGFb1 protein a higher and time-dependent TGFb1 secretion rate was observed for constructs harbouring Pro25 compared with Arg25. This finding was confirmed by Western Blot showing an increased level of secreted TGFb1 in presence of Pro25. Intracellular staining of TGFb1 in stably transfected cells was more pronounced for Pro25. FLAG epitope staining upon transient transfection indicated perinuclear localization of TGFb1. Western Blot using intracellular proteins suggests higher TGFb1 amounts for Pro25 than for Arg25.

Conclusions: This study suggests possible mechanisms responsible for the clinically observed genetic variability in TGFB1 in relation to radiosensitivity. Based on the here presented studies with allele-specific constructs the Pro25 variant in the signal peptide which affects about 10-15% of the European population might alter TGFb1 distribution leading to higher secretion rates in response to irradiation. The generated constructs will be used to further clarify the exact mechanisms of TGFb1 trafficking. If so, strategies to interfere with these processes could be developed with the aim to prevent patients at particular risk for radiation injuries and might be beneficial also for other conditions linked to TGFb1 signaling like chronic inflammations.

Personalized Laboratory Medicine

Brandslund Ivan, Pazzagli Mario, Vermeersch Pieter, Kerb Reinhold, Marc Janja

An EFLM/ESPT working group

The rapid developments of technology and methodology in the laboratory sciences have enabled mapping of the blue-print, pathways and processes in the human body in an unforeseen scale.

The results within the OMICS have clarified that not only are all healthy people different, but their diseases are as well.

The Pret-a-Porter approach to drug prescription, one size fits all, are no longer valid. And this is not only true for drugs, but also for other interventions as e.g. surgery, where intervention may be judged unnecessary if the right tools to predict the natural course of the disease were available (e.g. as in cervical neoplasia or breast cancer).

The prerequisite and absolute necessity for practicing personalized medicine is mastering personalized laboratory medicine.

The next few years will present tremendous opportunities, but also challenges, to the laboratory community to stand up and deliver both the right and relevant tests and volume of data and information with translation of these data to clinicians and patients.

Only if we can combine the production of genetic, metabolic and biomarker data with medical interventions, drugs and procedures using validated computer pattern recognition programs, yielding reliable predictions, can this challenge be met.

The working group has chosen breast-cancer as a model disease to develop a framework for deciding how to use which test in the patient-focused management of this disease. By the end of the year a position paper will be written.

We hope for contributions of knowledge from the scientific, laboratory and medical community to perform this task.

Population analysis and functional genetic polymorphisms of CYP2D6*4 and CYP2C19*17 cytochrome P450 and C3435T MDR1 gene

da Cunha Andrea, Dinis-Oliveira Ricardo, Queirós Odília, Moreira Roxana

Polymorphisms in genes encoding for xenobiotic metabolizing enzymes, such as the CYP2D6 and CYP2C19 isoforms of the cytochrome P450 (CYP450), and in genes encoding for proteins that mediate their efflux, such as the MDR1 gene encoding P-glycoprotein (Pgp), may affect the efficacy of drug therapy, determining the interindividual variability in drug resistance. In this context, and since the genes that encode for these proteins are highly polymorphic, it is necessary to perform detailed genetic and functional analyses.

Aiming to predict the incidence of certain genetic variations in the Portuguese population, the polymorphisms CYP2D6*4 and CYP2C19*17 (CYP450) and C3435T (MDR1 gene) have been studied in samples from volunteer donors of Instituto Superior de Ciências da Saúde-Norte, by PCR-RFLP and HRM assays. The frequency of the homozygous mutant genotype was the 7,4%, 7,7% and 13% for the CYP2C19*17, CYP2D6*4 and C3435T polymorphisms, respectively. To complement the population study, C3435T polymorphism was also genotyped in adenocarcinoma cell lines, HCT-15, HT29-MTX and Caco-2, in order to determine the influence of the polymorphism in Pgp functionality. Thus, cytotoxicity assays were performed using the antitumor drug doxorubicin, enabling to assess a possible relationship between the C3435T polymorphism and the corresponding metabolic profiles. In HCT-15, carrying a TT genotype, an increased resistance to the drug was found, comparing to the other cell lines, with CT genotype. It was also observed that HCT-15 cells showed a higher expression of Pgp, which was induced after exposure to the drug, being associated with the presence of the C3435T polymorphism.

Association of LIGHT polymorphism with response to warfarin therapy in Brazilian individuals

Bassani Borges Jessica, Dominguez Crespo Hirata, Danilo Cerda Maureira Alvaro, Moreno Fajardo Cristina, Reinel de Castro Lara, Ferraz Sampaio Marcelo, Cassilla dos Santos Jessica, Lin Wang Hui-Tzu, Helena Ortiz Lima Paula, Kenji Uemoto Toller Correia Gabriel, Marçal Dietzius Andrea, Nascimento Barbosa Oliveira Caroline, Guerra de Moares Rego Sousa Amanda, Dominguez Crespo Hirata Rosario, Hiroyuki Hirata Mario

Background: Nowadays warfarin is one of the most used oral anticoagulants and is affected by a broad interindividual variability. Environmental and genetic factors explain approximately half of the of warfarin dose requirements. Polymorphisms in genes associated with warfarin pharmacokinetics and pharmacodynamics have been extensively studied, however the influence of inflammation related genes, such as MMP9, LIGHT and LTA on warfarin response remains to be evaluated.

Objectives: To investigate the influence of MMP9, LIGHT and LTA polymorphisms on warfarin dose requirements in a cohort of Brazilian individuals.

Methods: Two hundred eighty seven patients (144 males and 143 females, aged 20-94 years) under warfarin therapy were selected from the Dante Pazzanese Institute of Cardiology, Sao Paulo, Brazil after Ethics Committee approval. Clinical data including INR values were recorded and blood samples were obtained to extract genomic DNA using the DNA blood mini kit (Qiagen, MD, USA).

The polymorphisms of MMP9, LIGHT and LTA were genotyped by pyrosequencing using PyroMark Q24®. Univariate and multiple logistic regression analyses adjusted for relevant covariates were carried out in order to evaluate the influence of clinical variables and genotypes on warfarin dose and time for achieving a stable INR. The patients were divided into quartiles according to dose at goal INR range and time to reach the first stable INR range.

Results: Minor allele frequencies for MMP9 rs17576 A>G (0.34), LIGHT rs2291668 C>T (0.16) and rs344560 G>A (0.07), LTA rs1041981 C>A (0.36) and rs909253 T>C (0.36) variants in our sample were similar to that available in 1000 Genome Project.

Univariate logistic regression showed that clinical variables such as gender, ethnics, hypertension, chronic renal insufficiency, cardiac insufficiency, dyslipidemia, acute myocardial infarction were not associated with variation in warfarin dose at goal INR range and time to reach the first stable INR range (p>0.05). Shortened time to reach the target INR was inversely related to the use of concomitant medications that increase (OR: 0.44, 95%CI: 0.21-0.936, p=0.033) or decrease the INR (OR: 0.25, 95%CI: 0.08-0.729, p=0.011). The LIGHT rs344560 A allele was associated with a lower weekly dose of warfarin (OR: 2.775, 95%CI: 1.01-7.63, p=0.002), however this association was not shown in the multivariate logistic regression when adjusted for the covariates age, gender, ethnics and target INR (OR: 2.543, 95%CI: 0.82-7.86, p=0.105). MMP9 and LTA polymorphisms studied were not associated with warfarin response.

Conclusions: LIGHT rs344560 variant may contribute to the variability of the warfarin dose requirement in anticoagulated patients and the time to reach the stable INR range is influenced by medications that alter the INR.

Contribution of polymorphisms in VKORC1, CYP2C9 and other candidate genes on therapeutic management of warfarin

Dominguez Crespo Hirata Thiago, Bassani Borges Jessica, Reinel de Castro Lara, Ferraz Sampaio Marcelo, Moreno Fajardo Cristina, Danilo Cerda Maureira Alvaro, Lin Wang Hui-Tzu lin, Cassilla dos Santos Jessica, Helena Ortiz Lima Paula, Kenji Uemoto Toller Correia Gabriel, Marçal Dietzius Andrea,Nascimento Barbosa Oliveira Caroline, Guerra de Moares Rego Sousa Amanda,Hiroyuki Hirata Mario,Dominguez Crespo Hirata Rosario

Background: Genetic variations can affect significantly the response to warfarin and consequently the dose requirements for adequate anticoagulation. The therapy is monitored by the international normalization ratio (INR) and clinicians try to maintain it within a narrow therapeutic range. Warfarin treatment management is challenging and essential to assure high efficacy with low risk of bleeding. It has been shown that environmental factors and polymorphisms in VKORC1, CYP2C9 and CYP4F2 contribute to variability of warfarin weekly dose. However the contribution of other genetic factors remains to be evaluated to better guide a personalized medicine.

Objectives: To evaluate the influence of VKORC1 and CYP2C9 as well as CYP2C19, CYP3A5, ABCB1, P2RY12, PTGS1, TBXAS1 polymorphisms on warfarin therapeutic management in a Brazilian population. Methods: Two hundred ninety patients (145 females and males, aged 13-94 years) under warfarin therapy were selected from the Dante Pazzanese Institute of Cardiology, Sao Paulo, Brazil. Clinical data were recorded and DNA was extracted. VKORC1, CYP2C9, CYP2C19, CYP3A5, ABCB1, P2RY12, PTGS1, TBXAS1 polymorphisms were identified by TaqMan® real time PCR. The influence of clinical variables and genotypes on warfarin dose and time both needed to reach the patient’s target INR were evaluated by univariate and multiple logistic regression analysis adjusted for relevant covariates. Results: Minor allele frequencies in Brazilian population for VKORC1 (rs9923231, rs7294, rs9934438), CYP2C9 (*2 allele, rs1799853), CYP2C9 (*3 allele, rs1057910), CYP2C19 (*2 allele, rs4244285), CYP3A5 (rs776746), ABCB1 (rs1045642), P2RY12 (rs2046934), PTGS1 (rs1236913), TBXAS1 (rs4725563), polymorphisms were 0.41, 0.36, 0.41, 0.11, 0.06, 0.15, 0.45, 0.37, 0.13, 0.07, 0.23 respectively. Clinical variables such as gender, ethnics and hypertension, chronic renal or cardiac insufficiency, dyslipidemia, acute myocardial infarction were not associated with variation in warfarin dose at goal INR range and time to reach the first stable INR range. Higher weekly dose of warfarin was associated with the VKORC1 rs7294 A allele (OR: 5.65, 95%CI: 1.92-16.60, p=0.002). On the other hand, lower weekly warfarin dose was related to VKORC1 rs9934438 A allele (OR: 8.33, 95%CI: 3.55-19.60), VKORC1 rs9923231 T allele (OR: 10.42, 95%CI: 4.24-25.58), CYP2C9*2 (OR: 2.06, 95%CI: 1.06-3.99) and CYP2C9*3 (OR: 8.06, 95%CI: 1.92-33.87), all p<0.05. Multivariate logistic regression analysis confirmed these results, except for CYP2C9*3. CYP3A5 rs776746 G allele was associated with less time to reach target INR (OR: 4.5, 95%CI: 1.46-14.42, p=0.009). CYP2C19, ABCB1, P2RY12, PTGS1 and TBXAS1 variants did not influence warfarin dose or time to reach target INR in this sample.

Conclusions: Polymorphisms in VKORC1 and CYP2C9 are the major contributors to the warfarin dose requirements, while the CYP3A5 rs776746 variant influences the time to reach target INR. Therefore these polymorphisms are relevant pharmacogenetic biomarkers for management of warfarin therapy in the Brazilian population.

No association between ERCC2 Lys751Gln polymorphism and colorectal cancer risk in an Algerian population

Moghtit F. Z., Louhibil, Jacques R, Le Morvan V, Bellot R, Boushaba A, Megaiz A, Bensenouci S, Boubekeur A, Abdelrhamane R, Mehtar N, Aberkane M

Colorectal cancer (CRC) is one of the most common causes of death due to cancer in both men and women throughout the world. It is a complex, multifactorial disease influenced by genetic and environmental factors. Genetic variation like Single Nucleotide Polymorphisms (SNP) in candidate genes such as DNA repair genes are thought to play an important role in individual variation in colorectal cancer susceptibility and in response to therapy. Many epidemiological studies have explored the association between ERCC2 (excision repair cross-complementing in rodents 2) polymorphisms and colorectal cancer risk in various populations. The aim of this case control study was to investigate the effect of ERCC2 A35931C (Lys751Gln, rs13181) polymorphism on the development of CRC among Algerian patients.

Genomic DNA was extracted from blood samples collected from 129 sporadic CRC patients and 148 normal controls. The Lys751Gln Polymorphism was determined by pyrosequencing technology.

The distribution of ERCC2 (Lys751Gln) genotype and allele frequencies did not differ significantly between CRC patients and healthy subject (P>0,05).

In conclusion, the results suggest that the Lys751Gln polymorphism of ERCC2 gene may not be associated with the colorectal cancer risk in Algerian population. Further research with a larger sample size is needed to reveal more information about this polymorphism and the appearance of colorectal cancer in our population

About the article

Published Online: 2013-08-01

Published in Print: 2013-08-01


These abstracts have been reproduced directly from the material supplied by the authors, without editorial alteration by the staff of this Journal. Insufficiencies of preparation, grammar, spelling, style, syntax, and usage are the authors’ responsibility.


Citation Information: Drug Metabolism and Drug Interactions, Volume 28, Issue 3, Pages A1–A48, ISSN (Online) 2191-0162, ISSN (Print) 0792-5077, DOI: https://doi.org/10.1515/dmdi-2013-0041.

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