La farmacogenética como herramienta de la medicina personalizada: desarrollo de estrategias para su implementación en la práctica clínica e identificación de nuevas asociaciones

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 28-03-2019La Farmacogenética (PGx) representa en la actualidad una de las herramientas más importantes del paradigma de la medicina personalizada permitiendo evitar Reacciones Adversas a Medicamentos (RAM) así como maximizar la eficacia de los tratamientos farmacológicos y, en definitiva, optimizar el proceso de selección de la mejor pauta terapéutica para cada paciente. El primer objetivo de esta tesis doctoral, fue el desarrollo de una estrategia para la implementación de la PGx en la rutina clínica de un hospital de primer nivel asistencial adscrito al Sistema Nacional de Salud (SNS). Esta estrategia está basada en una plataforma de microarray de SNPs que permite el genotipado simultáneo de 180 polimorfismos de respuesta a fármacos. A lo largo de este período (2013-2018) se han realizado un total de 3532 estudios PGx para distintos fármacos y en pacientes de distintas patologías dentro de la rutina clínica del hospital. Por otro lado, se evaluó la utilidad de un protocolo multidisciplinar para la implementación del genotipado anticipado de CYP2C19 en una cohorte de pacientes hematológicos previo al trasplante de progenitores hematopoyéticos, definida como población de riesgo para el tratamiento o profilaxis con Voriconazol. Esta estrategia permitió aumentar notablemente el porcentaje de pacientes que alcanzaban el rango terapéutico diana así como reducir el tiempo necesario para alcanzarlo, mejorando de este modo el éxito del tratamiento. El último objetivo de este trabajo fue la identificación de nuevas asociaciones PGx que permitiesen mejorar los algoritmos monogénicos existentes para la optimización del tratamiento con Tacrolimus y Voriconazol, mediante la incorporación de nuevos biomarcadores asociados a la variabilidad en su farmacocinética. A lo largo de esta tesis doctoral se proponen nuevos biomarcadores no incluidos hasta la fecha en los algoritmos clínicos que podrían resultar de interés para la individualización del tratamiento con estos fármacos. En definitiva, en este trabajo se ha desarrollado una estrategia que ha permitido la implementación de la PGx en la práctica clínica de nuestro centro. Además, se ha demostrado la utilidad en la práctica clínica de un protocolo de genotipado anticipado en una cohorte de pacientes hematológicos previo al trasplante de progenitores hematopoyéticos definida como población de riesgo para el tratamiento o profilaxis con Voriconazol. Finalmente, se proponen modelos poligénicos de predicción del perfil farmacocinético de dos fármacos de uso clínico frecuente, Voriconazol y Tacrolimus, que parecen mejorar los algortimos clínicos monogénicos desarrollados hasta la fecha.Pharmacogenetics (PGx) constitutes an essential tool for personalized medicine. It minimizes Adverse Drug Reactions (RAM) and maximizes treatment efficacy; and ultimately optimizes treatment selection for each patient. The first goal of this doctoral thesis was the development of a strategy for the implementation of PGx in the clinical routine of a third-level care hospital of the Spanish National Health Service. This strategy was based on a SNP-array platform allowing simultaneous genotyping of 180 SNPs related to drug response. During this period (2013-2018) we have performed 3532 PGx tests for different drugs and diseases in the clinical routine of our center. We have also evaluated the utility of a multidisciplinary protocol for the implementation of preemptive genotyping of CYP2C19 in a cohort of haematological patients, defined as risk population for treatment or prophylaxis with Voriconazole. This strategy resulted in a significant increase in the percentage of patients achieving Voriconazole goal therapeutic range and reduced the time window required to achieve it, and therefore improved treatment success. The last aim of this work was the identification of new PGx associations that improved the existing monogenic algorithms for Tacrolimus and Voriconazole treatment optimization, by the incorporation of new biomarkers related to their PGx interindividual variability. During this doctoral thesis we propose new biomarkers, not previously included in clinical prediction algorithms that may be of interest for treatment individualization with these drugs in the clinical practice. In summary, we have developed a strategy that allowed the implementation of PGx in the clinical routine of our center. In addition, we have demonstrated the clinical utility of a preemptive genotyping protocol in a cohort of haematological patients before allogenic hematopoietic cell transplantation defined as a risk population for treatment or prophylaxis with Voriconazole. Finally, we here propose two polygenic predictive models of the pharmacokinetic profile of Tacrolimus and Voriconazole that seem to improve the existing monogenic predictive algorithms

Synergistic effect of antimetabolic and chemotherapy drugs in triple-negative breast cancer

The triple-negative breast cancer (TNBC) subtype comprises approximately 15% of all breast cancers and is associated with poor long-term outcomes. Classical chemotherapy remains the standard of treatment, with toxicity and resistance being major limitations. TNBC is a high metabolic group, and antimetabolic drugs are effective in inhibiting TNBC cell growth. We analyzed the combined effect of chemotherapy and antimetabolic drug combinations in MDA-MB-231, MDA-MB-468 and HCC1143 human TNBC cell lines. Cells were treated with each drug or with drug combinations at a range of concentrations to establish the half-maximal inhibitory concentrations (IC50). The dose-effects of each drug or drug combination were calculated, and the synergistic or antagonistic effects of drug combinations were defined. Chemotherapy and antimetabolic drugs exhibited growth inhibitory effects on TNBC cell lines. Antimetabolic drugs targeting the glycolysis pathway had a synergistic effect with chemotherapy drugs, and antiglycolysis drug combinations also had a synergistic effect. The use of these drug combinations could lead to new therapeutic strategies that reduce chemotherapy drug doses, decreasing their toxic effect, or that maintain the doses but enhance their efficacy by their synergistic effect with other drugsMaría I. Lumbreras-Herrera and Andrea Zapater-Moros are supported by Consejería de Educación e Investigación de la Comunidad de Madrid (IND2018/BMD-9262). Elena López-Camacho is supported by the Spanish Economy and Competitiveness Ministry (PTQ2018–009760). This work is supported by an unrestricted grant from Roch

Utility of CYP2D6 copy number variants as prognostic biomarker in localized anal squamous cell carcinoma

Background: Anal squamous cell carcinoma (ASCC) is an infrequent tumor whose treatment has not changed since the 1970s. The aim of this study is the identification of biomarkers allowing personalized treatments and improvement of therapeutic outcomes. Methods: Forty-six paraffin tumor samples from ASCC patients were analyzed by whole-exome sequencing. Copy number variants (CNVs) were identified and their relation to disease-free survival (DFS) was studied and validated in an independent retrospective cohort of 101 ASCC patients from the Multidisciplinary Spanish Digestive Cancer Group (GEMCAD). GEMCAD cohort proteomics allowed assessing the biological features of these tumors. Results: On the discovery cohort, the median age was 61 years old, 50% were males, stages I/II/III: 3 (7%)/16 (35%)/27 (58%), respectively, median DFS was 33 months, and overall survival was 45 months. Twenty-nine genes whose duplication was related to DFS were identified. The most representative was duplications of the CYP2D locus, including CYP2D6, CYP2D7P, and CYP2D8P genes. Patients with CYP2D6 CNV had worse DFS at 5 years than those with two CYP2D6 copies (21% vs. 84%; p <.0002, hazard ratio [HR], 5.8; 95% confidence interval [CI], 2.7–24.9). In the GEMCAD validation cohort, patients with CYP2D6 CNV also had worse DFS at 5 years (56% vs. 87%; p =.02, HR = 3.6; 95% CI, 1.1–5.7). Mitochondria and mitochondrial cell-cycle proteins were overexpressed in patients with CYP2D6 CNV. Conclusions: Tumor CYP2D6 CNV identified patients with a significantly worse DFS at 5 years among localized ASCC patients treated with 5-fluorouracil, mitomycin C, and radiotherapy. Proteomics pointed out mitochondria and mitochondrial cell-cycle genes as possible therapeutic targets for these high-risk patients. Plain Language Summary: Anal squamous cell carcinoma is an infrequent tumor whose treatment has not been changed since the 1970s. However, disease-free survival in late staged tumors is between 40% and 70%. The presence of an alteration in the number of copies of CYP2D6 gene is a biomarker of worse disease-free survival. The analysis of the proteins in these high-risk patients pointed out mitochondria and mitochondrial cell-cycle genes as possible therapeutic targets. Therefore, the determination of the number of copies of CYP2D6 allows the identification of anal squamous carcinoma patients with a high-risk of relapse that could be redirected to a clinical trial. Additionally, this study may be useful to suggest new treatment strategies to increase current therapy efficacyIdiPAZ, Grant/Award Number: Jesús Antolín Garciarena Fellowship; European Proteomics Infrastructure Consortium, Grant/Award Number: 823839, Horizon 2020 Programm

MRX93 syndrome (BRWD3 gene): five new patients with novel mutations

Overgrowth syndromes (OGS) comprise a heterogeneous group of disorders whose main characteristic is that either the weight, height, or head circumference are above the 97th centile or 2 to 3 SD above the mean for age and sex. Additional features, such as facial dysmorphism, developmental delay or intellectual disability (ID), congenital anomalies, neurological problems and an increased risk of neoplasia are usually associated with OGS. Genetic analysis in patients with overlapping clinical features is essential, to distinguish between two or more similar conditions, and to provide appropriate genetic counseling and recommendations for follow up. In the present paper, we report five new patients (from four unrelated families) with an X-linked mental retardation syndrome with overgrowth (XMR93 syndrome), also known as XLID-BRWD3-related syndrome. The main features of these patients include ID, macrocephaly and dysmorphic facial features. XMR93 syndrome is a recently described disorder caused by mutations in the Bromodomain and WD-repeat domain-containing protein 3 (BRWD3) gene. This article underscores the importance of genetic screening by exome sequencing for patients with OGS and ID with unclear clinical diagnosis, and expands the number of reported individuals with XMR93 syndrome, highlighting the clinical features of this unusual disease.Instituto de Salud Carlos III, Grant/Award Number: PI15/01481.Peer reviewe

Molecular characterization of breast cancer cell response to metabolic drugs

Metabolic reprogramming is a hallmark of cancer. It has been described that breast cancer subtypes present metabolism differences and this fact enables the possibility of using metabolic inhibitors as targeted drugs in specific scenarios. In this study, breast cancer cell lines were treated with metformin and rapamycin, showing a heterogeneous response to treatment and leading to cell cycle disruption. The genetic causes and molecular effects of this differential response were characterized by means of SNP genotyping and mass spectrometry-based proteomics. Protein expression was analyzed using probabilistic graphical models, showing that treatments elicit various responses in some biological processes such as transcription. Moreover, flux balance analysis using protein expression values showed that predicted growth rates were comparable with cell viability measurements and suggesting an increase in reactive oxygen species response enzymes due to metformin treatment. In addition, a method to assess flux differences in whole pathways was proposed. Our results show that these diverse approaches provide complementary information and allow us to suggest hypotheses about the response to drugs that target metabolism and their mechanisms of action

Further delineation of neuropsychiatric findings in Tatton-Brown-Rahman syndrome due to disease-causing variants in DNMT3A: seven new patients

Tatton-Brown-Rahman (TBRS) syndrome is a recently described overgrowth syndrome caused by loss of function variants in the DNMT3A gene. This gene encodes for a DNA methyltransferase 3 alpha, which is involved in epigenetic regulation, especially during embryonic development. Somatic variants in DNMT3A have been widely studied in different types of tumors, including acute myeloid leukemia, hematopoietic, and lymphoid cancers. Germline gain-of-function variants in this gene have been recently implicated in microcephalic dwarfism. Common clinical features of patients with TBRS include tall stature, macrocephaly, intellectual disability (ID), and a distinctive facial appearance. Differential diagnosis of TBRS comprises Sotos, Weaver, and Malan Syndromes. The majority of these disorders present other clinical features with a high clinical overlap, making necessary a molecular confirmation of the clinical diagnosis. We here describe seven new patients with variants in DNMT3A, four of them with neuropsychiatric disorders, including schizophrenia and psychotic behavior. In addition, one of the patients has developed a brain tumor in adulthood. This patient has also cerebral atrophy, aggressive behavior, ID, and abnormal facial features. Clinical evaluation of this group of patients should include a complete neuropsychiatric assessment together with psychological support in order to detect and manage abnormal behaviors such as aggressiveness, impulsivity, and attention deficit-hyperactivity disorder. TBRS should be suspected in patients with overgrowth, ID, tall stature, and macrocephaly, who also have some neuropsychiatric disorders without any genetic defects in the commonest overgrowth disorders. Molecular confirmation in these patients is mandatory.This project was supported by ISCII, FEDER funds grant: FIS-PI15/01481

Clinical and molecular analyses of Beckwith-Wiedemann syndrome: Comparison between spontaneous conception and assisted reproduction techniques

Fil: Tenorio, Jair. Hospital Universitario La Paz. Instituto de Genética Médica y Molecular; Madrid, España.Fil: Romanelli, Valeria. Hospital Universitario La Paz. Instituto de Genética Médica y Molecular; Madrid, España.Fil: Martin-Trujillo, Alex. Centro de Investigación Biomédica en Red de Enfermedades Raras; Madrid, España.Fil: Fernández, García-Moya. Hospital Universitario La Paz. Instituto de Genética Médica y Molecular; Madrid, España.Fil: Segovia, Mabel. ANLIS Dr.C.G.Malbrán. Centro Nacional de Genética Médica; Argentina.Fil: Perandones, Claudia. ANLIS Dr.C.G.Malbrán. Centro Nacional de Genética Médica; Argentina.Fil: Pérez Jurado, Luis A. Universitat Pompeu Fabra; Barcelona, España.Fil: Esteller, Manel. Instituto de Investigación Biomédica de Bellvitge. Cancer Genetics Group; Barcelona, España.Fil: Fraga, Mario. Hospital Universitario Central de Asturias. Instituto Universitario de Oncología. Unidad de Epigenética del Cáncer; Oviedo, EspañaFil: Arias, Pedro. Hospital Universitario La Paz. Instituto de Genética Médica y Molecular; Madrid, España.Fil: Gordo, Gema. Hospital Universitario La Paz. Instituto de Genética Médica y Molecular; Madrid, España.Fil: Dapía, Irene. Hospital Universitario La Paz. Instituto de Genética Médica y Molecular; Madrid, España.Fil: Mena, Rocío. Hospital Universitario La Paz. Instituto de Genética Médica y Molecular; Madrid, España.Fil: Palomares, María. Hospital Universitario La Paz. Instituto de Genética Médica y Molecular; Madrid, España.Fil: Pérez de Nanclares, Guiomar. Hospital Universitario Araba. Molecular Genetics Laboratory. Research Unit; Vitoria-Gasteiz, España.Fil: Nevado, Julián. Hospital Universitario La Paz. Instituto de Genética Médica y Molecular; Madrid, España.Fil: García-Miñaur, Sixto. Hospital Universitario La Paz. Instituto de Genética Médica y Molecular; Madrid, España.Fil: Santos-Simarro, Fernando. Hospital Universitario La Paz. Instituto de Genética Médica y Molecular; Madrid, España.Fil: Martinez-Glez, Víctor. Hospital Universitario La Paz. Instituto de Genética Médica y Molecular; Madrid, España.Fil: Vallespín, Elena. Hospital Universitario La Paz. Instituto de Genética Médica y Molecular; Madrid, España.Fil: Monk, David. Instituto de Investigación Biomédica. Cancer Epigenetic and Biology Program. Imprinting and Cancer Group; Barcelona, España.Fil: Lapunzina, Pablo. Hospital Universitario La Paz. Instituto de Genética Médica y Molecular; Madrid, España.Beckwith-Wiedemann syndrome (BWS) is an overgrowth syndrome characterized by an excessive prenatal and postnatal growth, macrosomia, macroglossia, and hemihyperplasia. The molecular basis of this syndrome is complex and heterogeneous, involving genes located at 11p15.5. BWS is correlated with assisted reproductive techniques. BWS in individuals born following assisted reproductive techniques has been found to occur four to nine times higher compared to children with to BWS born after spontaneous conception. Here, we report a series of 187 patients with to BWS born either after assisted reproductive techniques or conceived naturally. Eighty-eight percent of BWS patients born via assisted reproductive techniques had hypomethylation of KCNQ1OT1:TSS-DMR in comparison with 49% for patients with BWS conceived naturally. None of the patients with BWS born via assisted reproductive techniques had hypermethylation of H19/IGF2:IG-DMR, neither CDKN1 C mutations nor patUPD11. We did not find differences in the frequency of multi-locus imprinting disturbances between groups. Patients with BWS born via assisted reproductive techniques had an increased frequency of advanced bone age, congenital heart disease, and decreased frequency of earlobe anomalies but these differences may be explained by the different molecular background compared to those with BWS and spontaneous fertilization. We conclude there is a correlation of the molecular etiology of BWS with the type of conception