Beate Beer

Association of polymorphisms in pharmacogenetic candidate genes (OPRD1, GAL, ABCB1, OPRM1) with opioid dependence in European population: a case-control study.

It is becoming increasingly evident that genetic variants contribute to the development of opioid addiction. An elucidation of these genetic factors is crucial for a better understanding of this chronic disease and may help to develop novel therapeutic strategies. In recent years, several candidate genes were implicated in opioid dependence. However, most study findings have not been replicated and additional studies are required before reported associations can be considered robust. Thus, the major objective of this study was to replicate earlier findings and to identify new genetic polymorphisms contributing to the individual susceptibility to opioid addiction, respectively. Therefore, a candidate gene association study was conducted including 142 well-phenotyped long-term opioid addicts undergoing opioid maintenance therapy and 142 well-matched healthy controls. In both study groups, 24 single nucleotide polymorphisms predominantly located in pharmacogenetic candidate genes have been genotyped using an accurate mass spectrometry based method. The most significant associations with opioid addiction (remaining significant after adjustment for multiple testing) were observed for the rs948854 SNP in the galanin gene (GAL, p = 0.001) and the rs2236861 SNP in the delta opioid receptor gene (OPRD1, p = 0.001). Moreover, an association of the ATP binding cassette transporter 1 (ABCB1) variant rs1045642 and the Mu Opioid receptor (OPRM1) variant rs9479757 with opioid addiction was observed. The present study provides further support for a contribution of GAL and OPRD1 variants to the development of opioid addiction. Furthermore, our results indicate a potential contribution of OPRM1 and ABCB1 SNPs to the development of this chronic relapsing disease. Therefore it seems important that these genes are addressed in further addiction related studies.

3D versus 2D cell culture implications for electron microscopy.

Cell culture systems are indispensable tools for basic research and a wide range of clinical in vitro studies. However, conventional 2D cell cultures poorly mimic the conditions in the living organism. This limitation may seriously compromise the reliability and significance of data obtained from such approaches. Therefore, we present here a comparative study on selected 3D and 2D cell cultures of U87-MG human glioblastoma cells that were processed by means of high-pressure freezing and freeze-substitution as well as by conventional chemical fixation and Tokuyasu cryo-section immuno-labeling. Three-dimensional cultures comprised pseudo-vascularized cultures, fiber and bead scaffold cultures, and spheroid cultures. Cell cultures in dishes and on coverslips were the static 2D culture systems used as reference models. We will discuss morphological and immuno-cytochemical observations with respect to the feasibility of the cell culture systems investigated for the state-of-the-art electron microscopy.

Adherence evaluation of endocrine treatment in breast cancer: methodological aspects

BackgroundCurrent studies on adherence to endocrine therapy in breast cancer patients suffer from methodological limitations due to a lack of well-validated methods for assessing adherence. There is no gold standard for measuring adherence. The aim of our study was to compare four different approaches for evaluating adherence to anastrozole therapy for breast cancer with regard to concordance between methods.MethodsOutpatients with early breast cancer treated with anastrozole completed the multi-method assessment of adherence. We implemented a self-report scale (the Simplified Medication Adherence Questionnaire), physician- ratings, refill records and determination of anastrozole serum concentration.ResultsComparison of the four approaches using Spearman rank correlation revealed poor concordance across all methods reflecting weak correlations of 0.2-0.4. Considering this data incomparability across methods, we still observed high adherence rates of 78%-98% across measures.ConclusionOur findings contribute to the growing body of knowledge on the impact that methodological aspects exert on the results of adherence measurement in breast cancer patients receiving endocrine treatment. Our findings suggest that the development and validation of instruments specific to patients receiving endocrine agents is imperative in order to arrive at a more accurate assessment and to subsequently obtain more precise estimates of adherence rates in this patient population.

CYP2D6 genotyping by liquid chromatography-electrospray ionization mass spectrometry

AbstractGenetic polymorphisms can significantly affect the enzyme activity of the drug metabolizing enzyme Cytochrome P450 2D6 (CYP2D6; OMIM 124030). Accordingly, CYP2D6 genotyping is considered as a valid approach to predict the individual CYP2D6 metabolizing status. We introduce ion-pair reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry (ICEMS) as method for the characterization of single base variants, small deletions, and insertions in the CYP2D6 gene. A two-step polymerase chain reaction (PCR) was developed for the simultaneous amplification of nine polymorphic regions within the CYP2D6 gene. Cleanup, separation, and denaturation of PCR amplicons were achieved by high-performance liquid chromatography. High-performance molecular mass measurements provided nucleotide composition profiles that principally enable the resolution of 37 reported CYP2D6 alleles. The developed assay was applied to the genotyping of 93 unrelated Austrian individuals. For validation, a selected number of samples and polymorphic sites were retyped by alternative genotyping technologies. The PCR-ICEMS assay turned out to be an accurate, robust, and cost-effective CYP2D6 genotyping strategy. FigureOutline of the principal steps of the PCR-ICEMS assay developed for CYP2D6 genotyping. A two-step PCR is used for the simultaneous amplification of nine polymorphic regions within the CYP2D6 gene. PCR amplicons are analyzed by ion-pair reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry (ICEMS). High-performance molecular mass measurements provide nucleotide composition profiles that enable the identification of CYP2D6 alleles

Optimized Conformal Paraaortic Lymph Node Irradiation is not Associated with Enhanced Renal Toxicity

Background and Purpose:For patients with gynecologic carcinomas, irradiation of paraaortic lymph nodes (PLNs) is a routine treatment concept. Planning target volumes (PTVs) individually optimized by radiation field delineations along the big vessels permit the inclusion of at least 97% of potentially involved PLNs. However, this novel treatment technique might increase radiation-induced nephrotoxicity. Therefore, the actual incidence of kidney damage after PLN irradiation has to be assessed in order to validate the safety of this treatment concept.Patients and Methods:19 patients were treated with irradiation alone (50.4 Gy; 5 × 1.8 Gy/week) and monitored for up to 90 months. Functional renal parameters, namely renal plasma flow (RPF) and glomerular filtration rate (GFR), were assessed by dynamic renal scintigraphy. Additionally, patients were clinically observed (i.e., hypertension, proteinuria) and calculations of normal-tissue complication probability (NTCP) values for nonuniform kidney irradiation were performed using the Lyman-Wolbarst algorithm.Results:Two patients with anticipated moderate NTCP values (12.6% and 8.7%) showed slightly impaired RPF rates at 12, 24, and after 48 months of follow-up. Only one patient in the subgroup showing NTCP values > 50% (n = 9) developed a notable impairment of renal RPF. However, all patients including those with elevated complication probabilities exhibited neither impaired GFR nor clinically apparent symptoms related to a loss of functioning renal tissue from 12 to > 48 months post irradiation.Conclusion:Conformal irradiation of retroperitoneal lymph nodes with individual PTV delineation appears not to be associated with clinically relevant functional impairment of the kidneys.Hintergrund und Ziel:Die Behandlung von Patientinnen mit gynäkologischen Tumoren beinhaltet häufig die Bestrahlung der paraaortalen Lymphknoten (PLNs). Durch eine individuelle Anpassung des Planungszielvolumens (PTV) an den Verlauf der großen abdominalen Gefäße können mindestens 97% aller potentiell befallenen PLNs behandelt werden. Die dadurch z.T. vergrößerten PTVs könnten aber mit einer gesteigerten Inzidenz für eine radiogene Nephropathie einhergehen. Um die Sicherheit dieser neuen Bestrahlungstechnik zu überprüfen, wurde das tatsächliche Auftreten von radiogenen Nephropathien nach solchen PLN-Bestrahlungen untersucht.Patienten und Methodik:19 Patientinnen mit gynäkologischen Tumoren, die eine Bestrahlung der PLNs (50,4 Gy; 5 × 1,8 Gy/Woche) ohne Chemotherapie in der Klinik der Autoren erhielten, wurden bis zu 90 Monate nachbeobachtet. Mittels seitengetrennter Nierenclearence wurden renaler Plasmafluss (RPF) und glomeruläre Filtrationsrate (GFR) bestimmt und die Patientinnen regelmäßig klinisch untersucht (u.a. Blutdruck, Proteinurie). Außerdem wurden für jede Patientin aus den dreidimensionalen Bestrahlungsplänen für beide Nieren NTCP-Werte (Wahrscheinlichkeit von Normalgewebskomplikationen) nach dem Lyman-Wolbarst-Algorithmus berechnet.Ergebnisse:Zwei Patientinnen mit moderaten NTCP-Werten (12,6% und 8,7%) wiesen nach 12, 24 und 48 Monaten eine leichte Störung des RPF auf. Nur eine Patientin aus der Gruppe mit NTCP-Werten > 50% (n = 9) entwickelte eine ausgeprägtere Störung des RPF. Keine Patientin zeigte eine Störung der GFR oder klinische Symptome einer Nierenschädigung in der Zeit von 12 bis > 48 Monate nach der Bestrahlung.Schlussfolgerung:Die konformale Bestrahlung der PLNs mit einem individuellen, dem Verlauf der großen Gefäße angepassten PTV führte im eigenen Patientenkollektiv in keinem Fall zu einer klinisch relevanten radiogenen Nephropathie.

A fatal intoxication case involving topiramate

Topiramate belongs to a new group of anticonvulsive drugs primarily applied in treatment of epilepsy and in preventive therapy of migraines. Topiramate is structurally unrelated to other antiepileptic drugs and acts by multiple neurostabilizing mechanisms. However, the pharmacology of topiramate appears to be complex and some of its pharmacodynamic actions still remain to be elucidated. This case report documents a fatal intoxication involving topiramate. A 41-year old woman with a known history of psychiatric disorder was found unresponsive by her husband. Resuscitation efforts did not succeed and the woman was pronounced dead at the intensive care unit four hours later. At the scene, drug packages of topiramate, citalopram and flunitrazepam were found. Autopsy including histological examination revealed morphological signs of an acute intoxication and shock. A comprehensive toxicological analysis with GC-MS was performed on the deceaseds autopsy samples (femoral blood, bile, kidney, gastric content). The results revealed the presence of topiramate at a concentration of 49mg/L in the femoral blood sample, thus clearly exceeding the therapeutic range. Additionally, citalopram (0.85mg/L) and flunitrazepam in traces (<2μg/L) were detected in peripheral blood. Based on the autopsy findings and toxicological results, the cause of death was primarily attributed to an intoxication with topiramate in combination with citalopram.

A novel amplification strategy for genotyping with liquid chromatography-electrospray ionization mass spectrometry

Among numerous available genotyping techniques, mass spectrometry (MS) based methods play a major role in providing high quality genotype data at reasonable costs for research and diagnostics, e.g. for pharmacogenetic applications. Ion-pair reversed-phase liquid chromatography hyphenated to electrospray ionization time-of-flight MS (ICEMS) is, for example, a powerful instrument that allows a direct characterization of complex mixtures of polymerase chain reaction (PCR) amplified DNA fragments. Current limitations of PCR-ICEMS genotyping are mainly concerned with the multiplex PCR set-up. Assay development often requires time-consuming primer design and intensive optimization of PCR conditions. To overcome this restraint, a robust amplification strategy originally combined with arrayed primer extension genotyping was transferred and adapted to ICEMS genotyping. The modifications involved limitation of the primer length, application of two universal sequences and amplification with an appropriate DNA polymerase. To demonstrate the applicability of the novel amplification strategy for ICEMS, a 23-plex pharmacogenetic genotyping assay was developed. After slight optimization steps, an efficient and quantitatively balanced amplification of all targeted markers was achieved, resulting in a convenient characterization of the multiplexed PCR fragments with ICEMS. Expenditure of time, costs and hands-on work associated with assay design and optimization was dramatically lowered compared to previous multiplex PCR-ICEMS assays. The developed 23-plex assay was applied in a pharmacogenetic study including 284 individuals (genotype call rate 99.0%). A total of 399 SNPs were retyped by Sanger sequencing (concordance rate 99.8%). The PCR-ICEMS assay turned out to be an accurate, reliable, cost-effective and a ready-to-use tool for pharmacogenetic genotyping.

Getting the Whole Picture: Adding Patient-reported Outcomes to Adjuvant Endocrine Treatment Evaluation in Premenopausal Breast Cancer Patients

To the Editor: The integration of Patient-Reported Outcomes (PROs) has gained relevance recently within treatment toxicity evaluation. Patient reports have been identified to provide complementary information on treatment toxicity (1,2) compared to proxy-rate based assessments (e.g., CTCAEs) in various cancer populations. This led the FDA and others (3,4) to recommend the utilization of PROs for cancer outcome research and in clinical routine. Looking at the well established, gold standard tamoxifen treatment (TT) for premenopausal, early breast cancer (BC), data on the so called excellent risk-benefit ratio (5) derived from the original admission trials at most lack PROs. At least, some more recent studies indicate a distinct prevalence of side effects originating from treatment induced estrogen suppression such as hot flushes or sexual problems. Understanding risks and benefits of TT from a patient perspective might thereafter be of vital importance for capturing a more precise picture of symptom burden in this patient population. Additionally, symptom burden is known to modulate adherence behavior particularly in patients who consider themselves as cured while experiencing iatrogenic harm. Regarding the considerable nonadherence rates reported in BC patients receiving tamoxifen (6,7) a detailed understanding of the association with TT toxicity might, thus, be essential for health the optimization of care efforts. The objective of our study was first to determine prevalence and severity of patient-reported physical side effects and psychosocial burden in premenopausal, early BC patients receiving upfront TT. Second, the degree of subjectively experienced symptoms compared with data derived from current pivotal trials was investigated. Additionally, the association between patient’s symptom burden and treatment adherence was determined. We conducted a computer-based, cross-sectional PRO-assessment (June 2009–February 2011) including the following instruments: FACT-B/+ES, HADS and the Self-reported measure of medication adherence questionnaire (SMAQ). Symptom frequencies were presented as percentages (95% confidence intervals) and compared with data from the ABCSG-12 trial (5). Ethical approval was provided for the study. The final study sample comprised 156 patients with a median age of 47 years (range 26–59) and a median TT duration of 18.3 months. 58.9% of all patients were fully adherent with regard to accuracy of medication intake according to the SMAQ. Most frequent symptoms were hot flushes (82.8%), sleep disorders (86.0%), and weight gain (39.7; please find details in Table 1). Overall, PROs indicated higher prevalence rates for all symptoms but depression as compared to data derived from the ABCSG-12 trial. Significantly higher PRO-prevalence rates were found for hot flushes, sleeping disorders, breast sensitivity, and fatigue. Symptoms significantly associated with TT adherence were vaginal itching/irritation (RR 1.54; 1.01–2.3), vaginal dryness (RR 1.51; 1.01–2.2), and weight gain (RR 1.78; 1.2–2.6). Vaginal itching or dryness, hence, seemed to increase the probability for nonadherence by about 50%, while weight gain was identified as the strongest associated factor with nonadherence. All other items did not show statistical significance. Our PRO study confirms a notably high level of psychosocial burden and physical side effects in premenopausal BC patients undergoing adjuvant TT. Evidence published only recently seems to support these findings claiming higher frequencies of several Address correspondence and reprint requests to: Georg Goebel, PhD, Department of Medical Statistics, Informatics and Health Economics, Innsbruck Medical University, Schoepfstrasse 41, A-6020 Innsbruck, Austria, or e-mail: georg.goebel@i-med.ac.at Contributed equally.

Abstract P1-11-02: Psychological morbidity in breast cancer survivors: Prevalence rates and determinants

Background: The number of breast cancer survivors (BCS) is steadily increasing due to improved treatment options, early detection and younger age at diagnosis. Thus, it is increasingly important to determine and better understand the psychological outcome following a cancer diagnosis and treatment in long-term. This might contribute to meeting the long-term health care demands of cancer survivors. We aimed at investigating levels and determinants of anxiety and depression (AD) in BCS. Patients and Methods: We included BCS with a non-metastatic disease in the stage of after-care. AD was determined as part of a cross-sectional, comprehensive patient reported outcome (PRO) assessment (incl. Functional Assessment of Cancer Therapy-G/+B/+ES, Eating Disorder Examination-Questionnaire, Sexual Activity Questionnaire and Body Image Scale) using the Hospital Anxiety and Depression Scale (HADS). Prevalence rates of AD and sample characteristics are presented descriptively using percentages, means and standard deviations. Predictors of anxiety and depression are identified by means of regression analysis. Results: A final sample of 743 breast cancer survivors who were on average 2.9 years post diagnosis (range: 0.1-11.3 years) participated in the study. Mean patient age was 56.4a (SD 11.5a), 2/3 of patients were postmenopausal. 22.5% of patients reported clinically relevant levels of anxiety and 11.2% of depression. Older age (β=0.012, t=2.53, p Conclusion: A distinct proportion of BCS report clinically relevant, long-term psychological morbidity. Especially older BCS, experiencing higher levels of endocrine symptoms and reduced functional well-being, seem to be at risk for psychological morbidity. A routine PRO-screening for psychological morbidity including the assessment of associated risk factors in this patient population might contribute to the identification of those women in need for psychological/ psychiatric treatment and in conjunction, improve cancer care. Citation Format: Hubalek M, Sztankay M, Oberguggenberger A, Meraner V, Egle D, Mangweth-Matzek B, Beer B, Huber N, Sperner-Unterweger B. Psychological morbidity in breast cancer survivors: Prevalence rates and determinants. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P1-11-02.

CYP2D6 genotyping in breast cancer patients by liquid chromatography-electrospray ionization mass spectrometry

Abstract The application of cytochrome P450 2D6 (CYP2D6) genotyping to allow a personalized treatment approach for breast cancer patients undergoing endocrine therapy has been repeatedly discussed. However, the actual clinical relevance of the CYP2D6 genotype in the endocrine treatment of breast cancer still remains to be elucidated. A major prerequisite for the successful and valid evaluation of the CYP2D6 genotype with regard to its pharmacokinetic and clinical relevance is the availability of a comprehensive, accurate and cost-effective CYP2D6 genotyping strategy. Herein we present a CYP2D6 genotyping assay employing polymerase chain reaction (PCR)-ion pair reversed-phase high-performance liquid chromatography-electrospray ionization time-of-flight mass spectrometry (ICEMS). The genotyping strategy involves the simultaneous amplification of nine variable regions within the CYP2D6 gene by a two-step PCR protocol and the direct analysis of the generated PCR amplicons by ICEMS. The nucleotide composition profiles generated by ICEMS enable the differentiation of 37 of the 80 reported CYP2D6 alleles. The assay was applied to type the CYP2D6 gene in 199 Austrian individuals including 106 breast cancer patients undergoing tamoxifen treatment. The developed method turned out to be a highly applicable, robust and cost-effective approach, enabling an economical CYP2D6 testing for large patient cohorts.