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Responsabile Roberta Rolla
Settore di ricerca Patologia Clinica
Personale docente Giorgio Bellomo  Patrizia Zeppegno  Oscar Alabiso
Personale non-docente Patrizia Pergolini  Matteo Vidali  Silvia Meola  Paola Pollarolo  Alice Appiani  Rita Portalupi      
Obiettivi della ricerca 1) Estimating the cost of redundancy in molecular diagnostics: the case of activated protein C resistance and factor V Leiden.
Risultati ottenuti 1) BACKGROUND: The activated protein C resistance--sensitivity ratio in the presence of Factor V deficient plasma (APC-SR/Factor V) exhibits a high sensitivity for factor V Leiden mutation and has been proposed as the diagnostic approach of choice, as an alternative to genetic tests, to evaluate activated protein C resistance. A survey, including 4969 requests, was performed on the activity of a typical Molecular Diagnostics Laboratory in order to estimate the costs due to reagents, instrumentation and personnel.

METHODS: The global costs of three hypothetical diagnostic approaches were compared: (A) exclusive molecular test for FV Leiden; (B) APC-SR alone; (C) APC-SR and the exclusive confirmation of positive results with molecular test.

RESULTS: The global cost for each patient with the three approaches investigated were respectively 42.20 euros (A), 1.09 euros (B), and 433 euros (C). The cost for finding a patient with factor V Leiden mutation was 549.00 euros for A, 14.18 euros for B, and 56.32 euros for C. It was calculated that a decrease of 97.42% and 89.74% can be obtained using the approaches B and C, respectively. The difference in cost between B and C can be justified by the avoidance of false positive cases (6%) and by the impossibility of distinguishing homozygous from heterozygous patients using APC-SR exclusively (B).

CONCLUSIONS: In the case of suspected phenotype APC resistance, we suggest a laboratory approach, which provides the combined and sequential use of ProCGlobal/FV analysis and a subsequent genetic test for positive patients. 
Obiettivi della ricerca 2) Plasma fibrinogen levels and restenosis after primary percutaneous coronary intervention.
Risultati ottenuti 2) BACKGROUND: Plasma fibrinogen levels influence restenosis following elective percutaneous coronary intervention (PCI) for stable angina. It is unknown whether the same is true in the setting of primary PCI. The aim of the study was therefore to assess whether fibrinogen levels were associated to 6-month in-stent restenosis (ISR) in STEMI patients undergoing successful primary PCI.

METHODS: From January 2003 to October 2004, 267 patients were admitted to our Institution for STEMI and treated by primary PCI. Of these, 171 patients met the inclusion criteria and were enrolled in our study. Fibrinogen levels were assessed at admission, 12 h, 24 h, 48 h, 72 h following PCI and at discharge. Six-month angiographic follow-up was 100% complete.

RESULTS: Subjects with 6-month ISR showed higher fibrinogen levels than patients without ISR. Patients in the upper fibrinogen tertile showed a higher 6-month incidence of symptoms and/or inducible myocardial ischemia (27.1% vs. 7.1%, P = 0.006) and a larger late lumen loss (1.3 ± 0.8 vs. 1.0 ± 0.9 mm, P = 0.049). Logistic regression analysis demonstrated a significant and independent association between fibrinogen levels and ISR.

CONCLUSIONS: Our study suggests that increased plasma fibrinogen levels are related to ISR in STEMI patients undergoing primary PCI. Larger studies are warranted to assess the prognostic value of fibrinogen over harder end-points. 
Obiettivi della ricerca 3) Role of the laboratory in monitoring patients receiving dual antiplatelet therapy.
Risultati ottenuti 3) BACKGROUND: The increasing demand for therapeutic monitoring in patients receiving antiplatelet therapy has been paralleled by the development of instruments and tests whose clinical usefulness is still under debate. We devised a laboratory approach to detect patients with antiplatelet resistance at risk to develop thrombotic events.

METHODS: 180 patients, under aspirin and clopidogrel after angioplasty and stent implantation, were studied by PFA100® with collagen/epinephrine (CoEPI, cut-off 165s) cartridge and by Multiplate® using arachidonic acid (ASPItest, pos <862AUC), ADP (ADPtest, pos <417AUC) and collagen (COLtest, pos <607AUC).

RESULTS: Only 67 out of 173 patients with ASPI<862 displayed a prolonged CoEPI and up to 65 patients had normal CoEPI despite ASPI<300. Patients with ASPI<300 had significantly lower COL than patients with ASPI>300. 138 patients displaying ADP<417 had significantly lower COL than those with ADP>417. Association between COL and ADP remained after ASPI stratification: in patients with suboptimal (ASPI 300-892) or maximal (ASPI<300) response to aspirin, having ADP<417 (clopidogrel responsive) increased COL positivity respectively from 9.5% to 58.8% and from 47.6% to 82.7%.

CONCLUSIONS: A combination of specific tests may be useful in identifying higher-risk patients with poor compliance or drug-resistance who potentially may benefit from therapy change. 
Obiettivi della ricerca 4) Side effects associated with ultrarapid cytochrome P450 2D6 genotype among women with early stage breast cancer treated with tamoxifen.
Risultati ottenuti 4) BACKGROUND: The side effects of tamoxifen, a drug widely used for the treatment and the prevention of recurrence in patients with estrogen receptor positive breast cancers (ER+), have been reported in clinical trials, but to date no information is available on their possible association with an increased enzymatic activity of CYP2D6 (ultra-metabolizers, UMs). The aim of this study was therefore to evaluate the association between the presence of multiple functional CYP2D6 alleles and the occurrence of side effects.

METHODS: 61 women with ER+ breast cancer receiving tamoxifen monotherapy have been investigated in order to assess the relationships between CYP2D6 UM phenotype and side effects. Genotyping of 16 CYP2D6 polymorphisms was performed using a new DNA microarray technology.

RESULTS: A difference highly significant (41.2 % of difference, 95 % CI 6-61%, Fisher`s exact test, p = 0.030) between numbers of Ultrarapid Metabolizers patients (UM; high activity) with two or more adverse drug reactions to tamoxifen (7/9; 77.8 %), compared to number of Extensive Metabolizers (EM; normal activity), Intermediate Metabolizers (IM; reduced activity) and Poor Metabolizers (PM; no activity) with at least two side effects (19/52, 36.5 %) was detected.
Similar difference was also observed comparing the two groups (UM vs EM-IM-PM) for the number of side effects (median and inter quartile range, IQR: AM / EM / IM 1, IQR 0-2 vs. ULTRA 2, IQR 2-4; Mann-Whitney p = 0.005).

CONCLUSIONS: Our results suggest a new association between CYP2D6 gene duplication and side effects to tamoxifen, indicating a possible role of CYP2D6 in their occurrence. 
Obiettivi della ricerca 5) Venlafaxine and CYP2D6 in clinical practice: work in progress.
Risultati ottenuti 5) BACKGROUND: Venlafaxine (V) is a serotonin-norepinephrine inhibitor, mainly metabolized by CYP2D6 to its active metabolite O-desmethylvenlafaxine (ODV). Depending on CYP2D6 activity, patients may be identified as Poor (PM), Intermediate (IM), Extensive (EM) or Ultrarapid Metabolizers (UM). There is some evidence that a PM phenotype is associated with poor tolerance more often than an EM, while a UM patient would only respond to a greater dose of V. The goal of this study was to evaluate the impact of CYP2D6 phenotype on the efficacy of V XR in depressed patients.

METHODS: In an observational study, we recruited 47 patients, (M= 18, F= 29, aged 18-65 years) satisfying DSM-IV criteria for Major Depressive Episode and receiving treatment with V 75-300mg/die. After providing their written informed consent, all patients underwent blood sampling for CYP2D6 genotyping, which was performed with INFINITI CYP2D6 assay, which employs AutoGenomics proprietary film-based microarray technology and can identify 16 different alleles.

RESULTS: We expect to find out a correlation between CYP2D6-genotype, Venlafaxine dose and clinical response to treatment in a larger population.

CONCLUSIONS: We will investigate whether a pharmacogenetic test prior to treatment can be useful in clinical practice to detect a proper Venlafaxine dosage or to switch to a different drug. 
Collaborazioni in atto   
Comunicazioni a congressi •R. Rolla, M. Vidali, S. Meola, P. Pollarolo, M.R. Fanello, C. Nicolotti, L. Forti, G. Borra, F. D’Agostino, V. Rossi, O. Alabiso and G. Bellomo. Association between ultrarapid CYP2D6 polymorphisms and adverse drug reactions among women with breast cancer treated with tamoxifen. 2° Congress of the European Society of Predictive Medicine, 14-15 May Berlin 2011.

•R. Rolla, M. Vidali, S. Meola, P. Pollarolo, M.R. Fanello, C. Nicolotti, L. Forti, G. Borra, F. D’Agostino, V. Rossi, O. Alabiso and G. Bellomo. Association between ultrarapid CYP2D6 polymorphisms and adverse drug reactions among women with breast cancer treated with tamoxifen. IFCC-WorldLab 21st International Congress of Clinical Chemistry and Laboratory Medicine, 15-19 May Berlin 2011.

•Rolla R, Sulas MG, Bardone MB, Mairate E and Bellomo G. Hemolysis is a major cause of variability in insulin measurement during oral glucose tolerance test in children. IFCC-WorldLab 21st International Congress of Clinical Chemistry and Laboratory Medicine, 15-19 May Berlin 2011.

•R. Rolla, M. Vidali, S. Meola, P. Pollarolo, M.R. Fanello, C. Nicolotti, L. Forti, G. Borra, F. D’Agostino, V. Rossi, O. Alabiso and G. Bellomo. Association between ultrarapid CYP2D6 polymorphisms and adverse drug reactions among women with breast cancer treated with tamoxifen. Convegno AIOM, 13-14 maggio Stresa 2011.
 
Pubblicazioni
Estimating the cost of redundancy in molecular diagnostics: the case of activated protein C resistance and factor V Leiden.