ADVERTIMENT. Lʼaccés als continguts dʼaquesta tesi queda condicionat a lʼacceptació de les condicions dʼús establertes per la següent llicència Creative Commons: http://cat.creativecommons.org/?page_id=184 ADVERTENCIA. El acceso a los contenidos de esta tesis queda condicionado a la aceptación de las condiciones de uso establecidas por la siguiente licencia Creative Commons: http://es.creativecommons.org/blog/licencias/ WARNING. The access to the contents of this doctoral thesis it is limited to the acceptance of the use conditions set by the following Creative Commons license: https://creativecommons.org/licenses/?lang=en Improved and efficient therapy of acromegaly by implementation of a personalized and predictive algorithm including molecular and clinical information PhD thesis by: Joan Gil Ortega Thesis supervisors: Prof. Manel Puig Domingo Dr. Mireia Jordà Ramos Tutor: Prof. Manel Puig Domingo Doctoral Program in Medicine. Department of Medicine. 2020 Acknowledgments M’agradaria agrair amb aquestes línies a les persones que han fet possible aquesta tesis, tant directament com indirectament. D’aquest període de la meva vida m’emporto moltes i bones experiències, grans aprenentatges i fins i tot, alguna nova habilitat. Però les persones que he trobat i m’han acompanyat durant aquests anys han estat el més important al·licient i el principal record que m’enduc d’aquests anys. I si parlem de persones no puc evitar anomenar, per contradictori que sembli, una institució, l’IGTP i l’antic IMPPC. On la primera persona que vaig conèixer va ser la meva directora de tesi Mireia Jordà que m’ha guiat durant tot aquest procés amb passió i dedicació. També vull agrair al Prof. Manel Puig per deixar-me participar en aquesta recerca tant captivadora en una malaltia tan interessant. A més a més, al IGTP he fet grans amics que sé que m’acompanyaran durant uns quants anys després d’aquesta experiència. He d’agrair a la gent del EiN les hores compartides a la Núria, la meva mestra en el laboratori, a la Bea, la Mireia, l’Helena, la Irene i tota la resta d’estudiants que han anant desfilant i que espero que els he ensenyat hagi estat profitós (especialment les minions Anna i Melania). A la gent del MAPlab i aniling, en especial a la Júlia, la Mar i la Berta per l’ajuda en moment crítics. També a les persones que he conegut dinant i que hem compartit molts dies i moltes històries, en Roberto, l’Emili, l’Izaskun, la Natàlia... Els qui m’han transmès l’amor per la bicicleta: l’Iñaki, en Sergio, en Lloyd i la Tanya amb el seu taller. I com no agrair als tres mosqueters: Edu, Laura i Marc els bons moments viscuts, molts que s’han gaudit i patit alhora. A la gent d’escalada: la Carme i la Marta. I fora de la recerca he d’agrair a la gent de la música també el suportar-me en molts moments. A les nenes del conservatori: la Vero, la Luisa, l’Ana i la Glòria. A l’Òria per fer-me de psicòloga de tant en tant. I a la meva estimada JOC que ha vegades m’ha fet anar més de cul que una altra cosa però que molts cops ha estat necessària. També voldria agrair a la meva família el seu suport en tot moment malgrat les vicissituds viscudes, en especial a la meva mare ja que aguantar a un fill fent el doctorat no és fàcil i els meus avis. Als meus professors de tots aquests anys, especialment a en Pep Lloreta, i a la gent del PRBB. I finalment, als amics de la UPF que alguns ja han passat pel tràngol del doctorat en especial a la Natàlia i a la Mónica. Moltes gràcies a tots. 1 Abbreviations ACC accuracy ACTH adrenocorticotropic hormone; also adrenocorticotropin or corticotropin AIP aryl hydrocarbon receptor interacting protein ARRB1 arrestin-beta 1 ARRB2 arrestin-beta 2 ATG autogel AUC area under the curve BMI body mass index cAMP cyclic adenosine monophosphate CDH1 E-cadherin; cadherin 1 CDH2 N-cadherin; cadherin 2 cDNA complementary DNA CI confidence interval CpG cytosine nucleotide is followed by a guanine nucleotide in the linear sequence CR complete responders to SRLs chorionic somatomammotropin hormone 1; also known as human placental CSH1 lactogen CSH2 chorionic somatomammotropin hormone 2 CSHL1 chorionic somatomammotropin hormone like 1 CV coefficient of variation DA dopamine agonists DNA deoxyribonucleic acid DRD1 dopamine receptor D1 DRD2 dopamine receptor D2 DRD5 dopamine receptor D5 EMT epithelial-mesenchymal transition ERK1 mitogen-activated protein kinase 3, extracellular signal-regulated kinase 1 ERK2 mitogen-activated protein kinase 1, extracellular signal-regulated kinase 2 ESRP1 epithelial splicing regulator 1 FC fold change 2 FFAs free fatty acids FIPA familial isolated pituitary adenoma FSH follicle-stimulating hormone GH growth hormone or somatotropin GH1 growth hormone 1; pituitary growth hormone GH2 growth hormone 2 GHR growth hormone receptor GHRH growth hormone-releasing hormone, also known as somatocrinin GHRHR growth-hormone-releasing hormone receptor GHRL ghrelin GHSR1a growth hormone secretagogue receptor 1A guanine nucleotide binding protein (G protein), alpha stimulating activity GNAS polypeptide 1 GUSB glucuronidase beta HBP high blood pressure HPRT1 hypoxanthine phosphoribosyl transferase 1 IGF-1 insulin-like growth factor 1, also called somatomedin C IHC immunohistochemistry In1-GHRL intron 1 ghrelin IRS-1 insulin receptor substrate 1 JAK2 Janus kinase 2 KLK10 kallikrein 10 LAR long acting release LH luteinizing hormone, also known as lutropin MAPK Mitogen-Activated Protein Kinases MEK Dual specificity mitogen-activated protein kinase kinase miRNA micro RNAs MRI magnetic resonance imaging MRPL19 mitochondrial ribosomal protein L19 MSH melanocyte stimulating hormone NeuroD4 neuronal differentiation 4 NR non-responders to SRLs OGTT oral glucose tolerance test OR odds ratio 3 PCR polymerase chain reaction phosphatidylethanolamine binding protein 1, also known as raf kinase inhibitory PEBPB1 protein PEG polyethylene glycol PGK1 phosphoglycerate kinase 1 pituitary-specific positive transcription factor 1; POU domain, class 1, transcription Pit1 factor 1 PLAGL1 pleiomorphic adenoma gene-like 1, also known as zinc finger 1 (ZAC1) PR partial responders to SRLs PRL prolactin, also known as lactotropin PSMC4 proteasome 26S subunit ATPase 4 qPCR quantitative polymerase chain reactions, also known as real time PCR REMAH Registro Molecular de Adenomas Hipofisarios phosphatidylethanolamine binding protein 1, also known as raf kinase inhibitory RKIP protein RNA ribonucleic acid ROC receiver operating characteristic RORC retinoic acid-related orphan receptor C RT-qPCR reverse trancription Qpcr SD standard deviation SDS standard deviation score SLRs somatostatin receptor ligands SNAI1 snail family transcriptional repressor 1 SNAI2 snail family transcriptional repressor 2 SRIF somatostatin; somatotropin release-inhibiting factor SST somatostatin sst5TMD4 splicing variant of SSTR5 with 4 transmembrane domains sst5TMD5 splicing variant of SSTR5 with 5 transmembrane domains SSTR1 somatostatin receptor 1 SSTR2 somatostatin receptor 2 SSTR3 somatostatin receptor 3 SSTR4 somatostatin receptor 4 SSTR5 somatostatin receptor 5 SSTRs somatostatin receptors 4 STAT1 signal transducer and activator of transcription 1 STAT5 signal transducer and activator of transcription 5 TBP TATA-Box Binding Protein TGF-β transforming growth factor beta TSH thyroid-stimulating hormone, also known as thyrotropin TWIST1 twist family bHLH transcription factor 1 VIM vimentin VIP secretin; vasoactive intestinal peptide 5 Contents Section Page Acknowledgments 1 Abbreviations 2 Abstract 8 Resum 9 1. Introduction 10 1.1 The Pituitary Gland 10 1.1.1 Anatomy and histology 10 1.1.2 Somatotroph cells and growth hormone 11 1.1.3 GH regulation 11 1.1.4 Peripheral GH actions 13 1.2 Acromegaly 14 1.2.1 History of acromegaly 14 1.2.2 Epidemiology 14 1.2.3 Pathogenesis 15 1.2.4 Diagnosis 16 1.2.5 Clinical Manifestations 17 1.3 Treatment of Acromegaly 18 1.3.1 Surgery 18 1.3.2 Radiation treatment 19 1.3.3 Dopamine agonist 19 1.3.4 Somatostatin receptor ligands (SRLs) 19 1.3.5 Pegvisomant 21 1.3.6 Choice of therapy 22 1.4 Clinical and molecular predictors to medical therapy response 23 1.5 Personalized medicine in acromegaly 27 2. Hypotheses 29 3. Objectives 30 4. Material and methods 31 4.1 Patients 32 4.2 Biochemical and hormonal assays 32 4.3 Bioethical statement 33 4.4 DNA and RNA isolation 33 4.5 Retrotranscription 33 4.6 Quantitative polymerase chain reaction 33 4.6 GNAS sequencing 35 4.7 E-cadherin promoter methylation assessment 35 4.8 Standard Statistical Analysis 38 4.9 Data mining analyses 39 5. Results 45 5.1 Study 1: Molecular profiling for acromegaly treatment: a validation study 46 5.2 Study 2: Association of Epithelial-mensenchymal transition (EMT) markers with response to somatostatin receptor ligands 6 in GH-secreting tumors 58 5.3 Study 3: Molecular determinants of enhanced response to somatostatin receptor ligands after debulking in large GH producing adenomas 66 5.4 Study 4: Data mining analyses for precision medicine in acromegaly 72 6. Discussion 81 7. Conclusions 91 8. Future perspective 92 9. Bibliography 95 10. Annex 118 10.1 Supplementary Tables 118 10.2 Supplementary Figures 122 7 Abstract Actual pharmacologic treatment in acromegaly is currently based upon assay-error strategy. The prompt biochemical control of the disease is essential to reduces comorbidities and mortality. Fortunately, several drugs have been developed over the years to treat acromegaly being first generation somatostatin receptor ligands (SRLs), the first-line treatment. However, up to 50% of patients do not respond adequately to SRLs, which delays biochemical control for months or even a year. The main objective of this thesis was to evaluate the potential usefulness of different molecular markers as predictors of response to SRLs and elaborate a new treatment algorithm accordingly. We taught advantage of the REMAH cohort of several nodes in Spain to collect 100 acromegaly samples and performed molecular analysis.