Morphofunctional analysis and identification of novel diagnostic and prognostic biomarkers of acute myocardial infarction and primary myelofibrosis

Abstract

During my PhD at the Laboratory of Human Anatomy and Biology applied to the hematopoietic system (SSD: BIO/16), I investigated the role and the diagnostic accuracy of platelet-based biomarkers in acute myocardial infarction, and the biological role and prognostic value of the rs1024611 SNP of CCL2 and the chemokine system CCL2/CCR2 in primary myelofibrosis. Acute myocardial infarction (AMI) represents one of the leading causes of morbidity and mortality worldwide. The main AMI clinical manifestation is chest pain, but often this symptom can be associated to different underlying pathological conditions other than AMI and only the 15% of chest pain patients generally receive a final diagnosis of AMI. Therefore, a timely diagnosis in chest pain patients, as well as a prompt and accurate risk stratification in patients with coronary disorders, represent an urgent and relevant clinical priority in both emergency and outpatient setting. Currently, the differential diagnosis of AMI is mainly based on a composite evaluation of patient medical history, physical examination, ECG, and biomarkers of myocardium necrosis. However, these biomarkers are detectable only after the acute event and are affected by several clinical factors. Here, I propose two different approaches, both based on platelets, to investigate the diagnostic performance of pre-existing biomarkers in blood circulation before the acute events or in the “peri-infarctual” period. Platelets are anucleate cells that play a key role in the initiation of atherosclerosis as well as in the final step of thrombus formation, considered the main biological processes underlying AMI. PKCɛ is a serine/threonine kinase exclusively expressed in platelets of AMI patients, in occurrence of the acute event. Here, I demonstrate that the evaluation of PKCɛ-expressing platelets by flow cytometry could be used in combination with the well established hs-cardiac troponin (cTn) to the diagnosis of AMI in patients with chest pain. Moreover, I evaluated platelet gene expression profile by microarray assay to identify a platelet gene signature specific for AMI. Indeed, platelets have a unique mRNA signature, mainly derived from their hematopoietic precursors. Therefore, gene expression profiling at the time of an AMI provides information concerning the platelet gene expression preceding the coronary event. Platelet transcriptome analysis using microarray identified five differentially expressed genes (DEGs) including FKBP5, S100P, SAMSN1, CLEC4E and S100A12. The logistic regression model based on these five DEGs show a very good diagnostic performance in discriminating ST-segment elevation myocardial infarction (STEMI) patients from both sCAD (patients with stable coronary artery disease) and healthy subjects, configuring the combination of these five DEGS as a promising biomarker for the diagnosis of STEMI.

During my PhD, I also studied the functional role of the rs1024611 polymorphism of CCL2 and the chemokine system CCL2/CCR2 in primary myelofibrosis. Primary myelofibrosis (PMF) is the most aggressive myeloproliferative neoplasm (MPN), representing the paradigm of onco-inflammation. Indeed, chronic inflammation, fuelled by neoplastic clone, contributes to several changes at both local (bone marrow fibrosis) and systemic levels (constitutional symptoms, pro-thrombotic state, second cancers). Inflammation in turn supports the expansion and evolution of the neoplastic clone, in a self-perpetuating vicious cycle. Cytokines are key mediators of this detrimental crosstalk between the neoplastic clone (primarily the megakaryocytic clone) and the bone marrow microenvironment. Among various cytokines, CCL2 is one of the most potent immune-modulatory and pro-fibrotic cytokines known to be elevated in PMF. CCL2 exerts its biological effects by preferentially binding to its receptor CCR2 and activating a downstream signaling which includes G-proteins, MAPK/ERK, PI3K/Akt and JAK/STAT pathways. CCL2 gene is highly polymorphic and SNPs in the regulatory regions of CCL2 account for the great inter-individual variability in CCL2 expression levels. Here, I demonstrate that male subjects homozygous (G/G) for the rs1024611 SNP of CCL2 have an increased risk of developing PMF and that, among patients with PMF, the G/G genotype is an independent prognostic factor for reduced overall survival. Concerning the functional role the SNP and the CCL2/CCR2 axis in PMF, I demonstrate that i) homozygous PMF patients are the highest CCL2 producers as compared to the other genotypes; ii) hemotopoietic progenitors (CD34+ cells) of PMF patients are a selective target of CCL2, since they uniquely express CCR2 (CCL2 receptor); iii) CCR2 expression correlates with bone marrow fibrosis in PMF, iv) the CCL2/CCR2 chemokine system boosts pro-survival signals via Akt phosphorylation; v) ruxolitinib effectively turns-off CCL2/CCR2 axis in PMF cells. Finally, the peculiar expression of CCR2 in CD34+ cells of PMF represents a promising diagnostic biomarker to discriminate patients with trueET and prePMF, as well as between prePMF and overtPMF. All the data reported in this thesis have been published on peer reviewed international scientific journals.