RESUMO
Background: Awareness about the importance of implementing DPYD pharmacogenetics in clinical practice to prevent severe side effects related to the use of fluoropyrimidines has been raised over the years. Since 2012 at the National Cancer Institute, CRO-Aviano (Italy), a diagnostic DPYD genotyping service was set up. Purpose: This study aims to describe the evolution of DPYD diagnostic activity at our center over the last 10 years as a case example of a successful introduction of pharmacogenetic testing in clinical practice. Methods: Data related to the diagnostic activity of in-and out-patients referred to our service between January 2012 and December 2022 were retrieved from the hospital database. Results: DPYD diagnostic activity at our center has greatly evolved over the years, shifting gradually from a post-toxicity to a pre-treatment approach. Development of pharmacogenetic guidelines by national and international consortia, genotyping, and IT technology evolution have impacted DPYD testing uptake in the clinics. Our participation in a large prospective implementation study (Ubiquitous Pharmacogenomics) increased health practitioners' and patients' awareness of pharmacogenetic matters and provided additional standardized infrastructures for genotyping and reporting. Nationwide test reimbursement together with recommendations by regulatory agencies in Europe and Italy in 2020 definitely changed the clinical practice guidelines of fluoropyrimidines prescription. A dramatic increase in the number of pre-treatment DPYD genotyping and in the coverage of new fluoropyrimidine prescriptions was noticed by the last year of observation (2022). Conclusion: The long path to a successful DPYD testing implementation in the clinical practice of a National Cancer Center in Italy demonstrated that the development of pharmacogenetic guidelines and genotyping infrastructure standardization as well as capillary training and education activity for all the potential stakeholders are fundamental. However, only national health politics of test reimbursement and clear recommendations by drug regulatory agencies will definitely move the field forward.
RESUMO
Resistance to 2,4-D (2,4-diclorophenoxyacetic acid) herbicide is increasing in various dicotyledonous weed species, including Papaver rhoeas, a weed infesting Southern European wheat crops. Non-target-site resistance to this herbicide is governed by a range of genes involved in herbicide stress response. To enable reliable measurement of gene expression levels in herbicide-resistant and susceptible plants it is necessary to normalize qPCR data using internal control genes with stable expression. In an attempt to find the best reference genes, the stability of seven candidate reference genes was assessed in plants resistant and susceptible to 2,4-D, subjected or not to herbicide stress. Using three statistical algorithms (geNorm, BestKeeper and NormFinder), the overall results revealed that glyceraldehyde-3-phosphate dehydrogenase, actin and ubiquitin were the most stable reference genes. The normalization expression levels of GH3 (indole-3-acetic acid amido synthetase) and GST3 (glutathione S-transferase) which are two genes up-regulated following 2,4-D treatment, were determined to verify the stability of these selected reference genes. A sudden increase in GH3 and GST3 expression was already detected 5h after herbicide application, confirming their involvement in plant response to 2,4-D. The validation results confirmed the applicability and accuracy of these reference genes. This study identified and validated reference genes in the non-model weed species P. rhoeas and these will facilitate gene expression analysis studies aimed at identifying functional genes associated with non-target-site resistance.